Identification of toxin ligands

ABSTRACT

The disclosure relates to a method and system of screening for ligands which specifically bind to receptors. The method comprises expressing at least one receptor. The at least one receptor is contacted with a sample comprising at least one ligand. Whether the ligand selectively binds to the receptor is determined.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/012,209, filed Dec. 7, 2007, and U.S.Provisional Patent Application Ser. No. 61/074,794, filed Jun. 23, 2008.The contents of the prior applications are herein incorporated byreference in their entireties.

ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT

Work for this invention was partially funded by a grant under GM54237awarded by the National Institute of Health. The government may havecertain rights in this invention.

BACKGROUND

Transmembrane proteins are key components of essential cellularfunctions. One particular class of transmembrane proteins, ion channels,are commonly characterized by the method utilized to open or close thechannel protein, either permitting or preventing specific ions frompermeating the channel protein and crossing the cellular membrane. Forexample, one type of channel protein is the voltage-gated channelprotein, which is opened or closed in response to changes in electricalpotential across the cell membrane. Another type of channel protein ismechanically gated, such that mechanical stress on the protein opens orcloses the channel. Still another type is ligand-gated, such that itopens or closes depending on whether a particular ligand is bound theprotein. The ligand can be either an extracellular moiety, such as aneurotransmitter, or an intracellular moiety, such as an ion ornucleotide.

Transmembrane proteins such as ion channels are involved in a widevariety of biological process, such as cardiac, skeletal, and smoothmuscle contraction, nerve function, epithelial transport of nutrientsand ions, T-cell activation and pancreatic beta-cell insulin release.For example, one common type of channel proteins, K⁺ ion channels,control heart rate, regulate the secretion of hormones such as insulininto the blood stream, generate electrical impulses central toinformation transfer in the nervous system, and control airway andvascular smooth muscle tone. Thus, K⁺ ion channels participate incellular control processes that are abnormal, such as cardiacarrhythmia, diabetes mellitus, seizure disorder, asthma andhypertension.

In the search for new drugs, diagnostics, or research tools,transmembrane proteins are therefore a common target.

SUMMARY

In one aspect, the present disclosure provides systems and methods foridentifying or detecting a ligand in a sample. Such systems and methodsinclude contacting at least one receptor with a sample comprising atleast one toxin peptide; and determining whether a toxin peptide in thesample selectively binds to the at least one receptor, therebyidentifying or detecting a ligand in a sample.

In some embodiments, the at least one receptor is expressed in cells,and the method includes transfecting the cells with a nucleic acidencoding the at least one receptor. The at least one receptor caninclude a receptor that is heterologous to the cell in which it isexpressed. The at least one receptor can include a receptor that isnative to the cell in which it is expressed. The at least one receptorcan include a receptor that is stably expressed in a cell.

In some embodiments, transfected cells include at least two cellscomprising a first cell expressing a first receptor and a second cellexpressing a second receptor different from the first receptor. In someembodiments, the at least one receptor is immobilized on a substrate.

The at least one receptor can include a transmembrane protein, e.g., achannel protein, e.g., a channel protein selected from the groupconsisting of a sodium ion channel, a potassium ion channel, a calciumion channel, a chloride ion channel, a non-specific ion channel. In someembodiments, a transmembrane protein includes a potassium ion channel.In some embodiments, a potassium ion channel is a Kv1.3 channel.

In some embodiments, a sample includes a library of toxin peptides,e.g., a phage display library. In some embodiments, toxin peptides areabout 5-200 amino acids in length. In some embodiments, toxin peptidesare about 5-100 amino acids in length. In some embodiments, toxinpeptides are about 5-50 amino acids in length. In some embodiments,toxin peptides are about 20-50 amino acids in length. In someembodiments, toxin peptides are about 30-50 amino acids in length. Toxinpeptides can include sequences found in a toxin that is naturallyexpressed in an organism (e.g., a snake toxin, a snail toxin, a scorpiontoxin, a sea anemone toxin, a spider toxin, a lizard toxin). In someembodiments, a toxin peptide includes six cysteine residues. In someembodiments, spacing of the cysteine residues is conserved with thespacing of cysteine residues found in a natural toxin. In someembodiments, a disulfide bonding pattern is conserved with a disulfidebonding pattern found in a natural toxin. For example, in someembodiments, a toxin peptide includes at least 35 amino acids, and hasan amino acid sequence including at least six cysteine residues, so thatthe cysteine residues are located at each of the following positionswithin the 35 amino acids: 7 or 8, 13 or 14, 27 or 28, 32 or, 33, and 34or 35. In some embodiments, a toxin peptide library includes toxinpeptides in which a disulfide bonding pattern is conserved with adisulfide bonding pattern found in a natural toxin, and in whichresidues other than cysteines are altered (e.g., by randomization ofresidues at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 positions of atoxin sequence).

In some embodiments, toxin peptides have an amino acid sequence thatallow variability at “X” residues, e.g., toxin peptides have an aminoacid sequence that includes:(X)_(m)C(X)_(n)C(X)_(n)C(X)_(o)C(X)_(n)CXC(X)_(m) (SEQ ID NO:______),wherein X is any amino acid, and wherein m=0-10 amino acids, n=2-10amino acids, and o=2-20 amino acids. In some embodiments, m is 2. Insome embodiments, m is 4. In some embodiments, m is 5. In someembodiments, m is 6. In some embodiments, n is 3. In some embodiments, nis 4. In some embodiments, o is 9. In some embodiments, o is 10. In someembodiments, o is 11.

In some embodiments, toxin peptides have an amino acid sequence thatincludes: (X)_(m)KC(X)_(n)QC(X)_(n)CK(X)_(o)KCM(X)_(n)CXC(X)_(m) (SEQ IDNO:______), wherein X is any amino acid, and wherein m=0-10 amino acids,n=2-10 amino acids, and o=2-20 amino acids. In some embodiments, toxinpeptides have an amino acid sequence that includes:XXXKCXXXXQCXXXCKXXXXXXXKCMXXXCXCXX (SEQ ID NO:______), wherein X is anyamino acid.

A toxin peptide library can include a plurality of unique toxinpeptides, e.g., at least 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹², or more unique toxin peptides.

The present disclosure also provides libraries of toxin peptides. Insome embodiments, a library of toxin peptides includes a plurality ofmembers having an amino acid sequence that includes:(X)_(m)C(X)_(n)C(X)_(n)C(X)_(o)C(X)_(n)CXC(X)_(m) (SEQ ID NO:______),wherein X is any amino acid, and wherein m=0-10 amino acids, n=2-10amino acids, and o=2-20 amino acids. In some embodiments, m=2-10 aminoacids, n=3-5 amino acids, and o=7-12 amino acids. In some embodiments, mis 2. In some embodiments, m is 4. In some embodiments, m is 5. In someembodiments, m is 6. In some embodiments, n is 3. In some embodiments, nis 4. In some embodiments, o is 9. In some embodiments, o is 10. In someembodiments, o is 11.

In some embodiments, a library of toxin peptides includes a plurality ofmembers having an amino acid sequence that includes:(X)_(m)KC(X)_(n)QC(X)_(n)CK(X)_(o)KCM(X)_(n)CXC(X)_(m) (SEQ IDNO:______), wherein X is any amino acid, and wherein m=0-10 amino acids,n=2-10 amino acids, and o=2-20 amino acids. In some embodiments, m=2-10amino acids, n=3-4 amino acids, and o=7-12 amino acids. In someembodiments, a library of toxin peptides includes a plurality of membershaving an amino acid sequence that includes:XXXKCXXXXQCXXXCKXXXXXXXKCMXXXCXCXX (SEQ ID NO:______), wherein X is anyamino acid.

In some embodiments, a library of toxin peptides includes toxin peptidesfrom one or more of a snake toxin, a snail toxin a scorpion toxin, a seaanemone toxin, and a lizard toxin.

The present disclosure also provides mokatoxin-1, mokatoxin-2, andmokatoxin-3 peptides, and variants thereof (e.g., variants thatspecifically bind to, and inhibit, a Kv1.3 channel). In someembodiments, the disclosure provides a peptide comprising an amino acidsequence that is at least 95% identical to SEQ ID NO: 1. In someembodiments, the disclosure provides a peptide comprising an amino acidsequence that is at least 95% identical to SEQ ID NO: 2. In someembodiments, the disclosure provides a peptide comprising an amino acidsequence that is at least 95% identical to SEQ ID NO: 3. Pharmaceuticalcompositions including the peptides and methods of using thecompositions, e.g., for immune suppression, are also provided herein.

In another aspect, the present disclosure provides a peptide comprisingthe following amino acid sequence:IXVKCXXPXQCXXPCXXXGXXXXXKCMNXKCXCYX_(n) (SEQ ID NO: ______), wherein Xis any amino acid, wherein n=1-20 amino acids, and wherein the peptidespecifically binds to a potassium channel.

In still another aspect, the present disclosure provides a peptidecomprising the following amino acid sequence:IXVKCXXPXQCXXPCKXXGXXXXKCMNXKCXCYX_(n) (SEQ ID NO: ______), wherein X isany amino acid, wherein n=1-20 amino acids, and wherein the peptidespecifically binds to a potassium channel.

Other systems, methods, features and advantages will be, or will become,apparent to one with skill in the art upon examination of the followingfigures and detailed description. It is intended that all suchadditional systems, methods, features and advantages be included withinthis description, be within the scope of the disclosure, and beprotected by the following claims.

BRIEF DESCRIPTION OF THE DRAWING

Provided methods and systems may be better understood with reference tothe following drawings and description. Components in the figures arenot necessarily to scale, emphasis instead being placed uponillustrating principles of the disclosure. In the figures, likereferenced numerals designate corresponding parts throughout thedifferent views.

FIG. 1 is a schematic representation of the partial amino acid sequenceof a ligand construct including animal venom neurotoxin kaliotoxin-1(KTX) in a phage display suitable vector.

FIG. 2 is a schematic representation of the partial amino acid sequencesof an animal toxin library.

FIG. 3 is a graph depicting the specific enrichment of KTX by Kv1.3transfected cells.

FIG. 4 is a set of graphs depicting the specific binding of differentanimal venom toxin phages to their respective targets quantified byELISA.

FIG. 5 is a set of graphs depicting the preferential selection of animalvenom toxin snake Dendroaspis natriuretic peptide (DNP) and snakeAtractaspis sarafotoxin S6b ligands in a phage display system onantiserum raised against DNP and S6b, respectively.

FIG. 6 is a set of graphs depicting KTX binding to KcsA carrying theKv1.3 pore domain (KcsA-1.3), quantified by ELISA.

FIG. 7 is a set of graphs depicting KTX-phage inhibition of Kv1.3channels expressed in HEK293 cells.

FIG. 8 is a schematic representation of one type of representativedesign of a combinatorial toxin library based on KTX and similar toxins.

FIG. 9 is a schematic representation of the novel amino acid sequencesof mokatoxin-1, mokatoxin_(—)0422, and mokatoxin_(—)0516, and controlsKTX and inactive KTX.

FIG. 10 is a graph depicting selective blockade of wild-type Kv1.3channels by mokatoxin-1 expressed in Xenopus oocytes.

FIG. 11A is a graph depicting mokatoxin_(—)0422-phage inhibition ofKv1.3 currents at 0.3 nM toxin-phage concentration.

FIG. 11B is a graph depicting mokatoxin_(—)0516-phage inhibition ofKv1.3 currents at 1.0 nM and mokatoxin_(—)0422-phage inhibition of Kv1.3currents at 1.0 nM toxin-phage concentration.

FIG. 12 is a set of graphs depicting pharmacologically activeDendroaspis dendrotoxin blocking Kv1.1 K⁺ channels in mammalian cellswhen expressed on the phage. Camel VHH, nonspecific-phage, and phagebuffer cause no block.

FIG. 13 is a graph depicting functional Shk expressed on the phageblocking Kv1.3 current in mammalian cells.

FIG. 14 is a table showing an alignment of peptides from toxins producedby various Conus species. The disulfide bonding pattern of the peptidesis indicated under each set.

FIG. 15 is a table showing the amino acid sequences of various toxinpeptides. Animal species, peptide length, receptor target, and positionsof disulfide bonds are also indicated.

FIG. 16 is a graph which shows the dose-response relationship ofmokatoxin-1 on different K channels: human (h) Kv1.1, Kv1.2, Kv1.3, andmouse (m) big conductance calcium-activated K channel.

FIG. 17 (A-C) is a set of graphs showing that treatment with MK toxininhibits the secretion of effector cytokines by T cells. 10⁵ purified Tcells were treated with MK and KTX toxin 1 h prior activation andstimulated for 16 h with CD3/CD28 beads at a 1:1 ratio. The supernatantwere assessed for IL-2 (C), TNF-α (A) and IFN-γ (B) secretion by ELISA.The graphs show the results of a representative experiment from 2independents assays. *, p<0.05; **, p<0.01.

FIGS. 18A and 18B are a set of graphs showing the selectivity ofmokatoxin-1 for Kv1.3 K⁺ channel subtype. (A) Kaliotoxin (KTX) at 10 nM,but not mokatoxin-1 (tested: 1-100 nM, shown: 100 nM), induced twitchesin the ileum strips. (B) The classical kaliotoxin homolog margatoxin(MgTX) at 10 nM, but not mokatoxin-1 (tested: 1-100 nM, shown: 100 nM),induced a lowering of the pressure threshold for initiation of theperistaltic waves and an increase the frequency of these waves.

FIG. 19 is a graph showing the kinetics of inhibition of hKv1.3 bymokatoxin-2.

FIG. 20 is a graph showing the kinetics of inhibition of hKv1.3 bymokatoxin-3.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present disclosure provides for systems for identifying,characterizing, and detecting ligands that bind to particular receptors(also referred to herein as targets), compositions including theligands, and methods of using the ligands. Methods of identifyingligands described herein permit selection of ligands that exhibit adesired degree of specificity, affinity, and/or biological activity fora target of interest. The present disclosure encompasses the discoveryof novel ligands (e.g., ligands identified from variegated libraries oftoxin peptides) having a high degree of selectivity for specificreceptors such as ion channels, as well as methods for producing andusing the ligands to modulate receptor activity. Exemplary ligandsdescribed herein include mokatoxin-1, mokatoxin-2, and mokatoxin-3, eachof which specifically bind to the receptor Kv1.3. Other ligands andreceptors are also described.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present disclosure. All publications, patentapplications, patents, references to amino acid and nucleic acidsequence database identifiers, and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

DEFINITIONS

As used herein, the term “characteristic sequence element” or “sequenceelement” refers to a stretch of contiguous amino acids, typically 5amino acids, e.g., at least 5-500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-15,or 5-10 amino acids, that shows at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identity with another polypeptide. In someembodiments, a characteristic sequence element participates in orconfers function on a polypeptide.

As used herein, the term “corresponding to” is often used to designatethe position/identity of an amino acid residue in a polypeptide (e.g.,in a toxin). Those of ordinary skill will appreciate that, for purposesof simplicity, a canonical numbering system (based on wild type toxins)is utilized herein (as illustrated, for example, in FIGS. 2, 9, 14, and15), so that an amino acid “corresponding to” a residue at position 7,for example, need not actually be the 7^(th) amino acid in a particularamino acid chain but rather corresponds to the residue found at 7 in awild type polypeptide (e.g., in a toxin); those of ordinary skill in theart readily appreciate how to identify corresponding amino acids.

The term “library” refers to a collection of members. A library may becomprised of any type of members. For example, in some embodiments, alibrary comprises a collection of phage particles. In some embodiments,a library comprises a collection of peptides. In some embodiments, alibrary comprises a collection of cells. A library typically includesdiverse members (i.e., members of a library differ from each other byvirtue of variability in an element, such as a peptide sequence, betweenmembers). For example, a library of phage particles can include phageparticles that express unique peptides. A library of peptides caninclude peptides having diverse sequences. A library can include, forexample, at least 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or moreunique members.

The term “ligand” refers to any agent that binds to a receptor. Ligandscan include, but are not limited to, small molecules (whether syntheticor isolated from natural sources), biodegradable cofactors, proteins,synthetic peptides, and polymers, both synthetic and naturallyoccurring, including DNA. In many embodiments, ligands are polypeptides.For example, ligands can be protein/peptide toxins and/or othervenom/poison components of animal, plant, or microbial origin or naturalor synthetic derivatives of the same. In some embodiments, a ligand is atoxin peptide as defined herein. In some embodiments, ligands areexpressed and/or presented to a receptor as part of a library, e.g., aphage display library. In some embodiments, ligands are expressed and/orpresented singly. A ligand can be presented to a receptor in any othermean or form (e.g., removed from the phage, or expressed in acomparable/other expression systems) suitable for ligand-targetselection and/or ligand validation. In some embodiments, a ligand is aphage-only peptide to monitor or alter a selection process. According tomethods described herein, ligands can be selected on any of a variety ofbases, including for example on the basis of a particular affinity,specificity, or activity toward a receptor of interest. In certainembodiments, a ligand binds to a receptor with a K_(D) of 1×10⁻⁶ M orless, 1×10⁻⁷ M or less, 1×10⁻⁸ M or less, 1×10⁻⁹ M or less, 1×10⁻¹⁰ M orless, 1×10⁻¹¹ M or less, or 1×10⁻¹² M or less. In certain embodiments, aligand binds a receptor which is a channel, and inhibits an activity ofthe channel (e.g., ion transport) with an IC₅₀ of 1×10⁻⁶ M or less,1×10⁻⁷ M or less, 1×10⁻⁸ M or less, 1×10⁻⁹ M or less, 1×10⁻¹⁰ M or less,1×10⁻¹¹ M or less, or 1×10⁻¹² M or less. In some embodiments, a ligandhas specificity a particular receptor such that the ligand binds to thereceptor and/or modulates an activity of the receptor with anaffinity/potency that is at least twice, 4 times, 5 times, 10 times, 100times, 1000 times as great as for another receptor in the same class. Togive one example, in some embodiments, a ligand binds to one type ofpotassium channel, Kv1.3, with an affinity that is at least 10 or 100times greater than its affinity for another potassium channel (e.g.,Kv1.1 or Kv1.2).

The term “receptor” (also referred to herein as a “target”) refers to amolecule, part of a molecule, chimera of more than one molecule or partsof it, or an assembly of molecules that serves as an interacting partnerfor a ligand. In some embodiments, a receptor is a receptor for a toxinpeptide. In some embodiments, a receptor is a channel polypeptide. Forexample, receptors for toxin peptides include, but are not limited to,Ca²⁺ channel, Na⁺ channels, K⁺ channels, NMDA receptor,alpha1-adrenoceptor, neurotensin receptor, Cl⁻ channel, noradrenalinetransporter, vasopressin receptor, acetylcholinesterase, endothelinreceptor, natriuretic peptide receptor, GPIIb/IIIa integrin receptor,muscle-type nicotinic acetylcholine receptor (nAChR), neuronal-typenAChR, muscarinic acetylcholine (ACh) receptor, serotonin (5-HT)receptor, angiotensin-converting enzyme. Antibodies and other specificmolecular partners for toxin peptides are also defined as receptors.Receptors may be wild-type receptors or natural or synthetic variants ofwild-type receptors. “Receptor” further refers to all protein families(types or superfamilies) that include at least one member (also known assubtype or isoform) that are receptors for toxin peptides. For example,a receptor subtype Kv2.1 K⁺ channel has no known toxin peptide ligand,but at least one other member of the K⁺ channel protein family (forexample subtype Kv1.3) has a toxin ligand and therefore, members of theK⁺ channel family in its entirety are considered “receptors.”

A “scaffold” is a structural element, or set of elements, that is commonto a set of structurally related compounds. For example, a toxin peptidescaffold may include one or more particular amino acids located atparticular positions along the polypeptide chain of a toxin. In someembodiments, a toxin peptide scaffold may include a specific number andarrangement of disulfide bridges. A scaffold can include a sequencehaving 50% or higher sequence identity with the toxin, or less that 50%identity if residues required for interaction with a receptor are thesame or similar. In some embodiments, two toxins are said to share ascaffold if the toxins include a similar number of amino acid residuesand cysteine residues found within the sequences having similar spacing.Alternatively or additionally, a toxin peptide scaffold may include oneor more amino acids found at particular positions within a sequenceelement (typically at least 5-20 amino acids long) found in a toxin. Insome embodiments, at least 50% of the amino acid residues in a givensequence element are scaffold amino acids. In some embodiments, at least55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or moreof the amino acids in a sequence element are scaffold amino acids.

The term “substantial identity” of amino acid sequences (and ofpolypeptides having these amino acid sequences) typically means sequenceidentity of at least 40% compared to a reference sequence as determinedby comparative techniques known in the art. For example, a variety ofcomputer software programs are well known for particular sequencecomparisons. In some embodiments, the BLAST is utilized, using standardparameters, as described. In some embodiments, the preferred percentidentity of amino acids can be any integer from 40% to 100%. In someembodiments, sequences are substantially identical if they show at least40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical residuesin corresponding positions. In some embodiments, polypeptides areconsidered to be “substantially identical” when they share amino acidsequences as noted above except that residue positions which are notidentical differ by conservative amino acid changes. Conservative aminoacid substitutions refer to the interchangeability of residues havingsimilar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, asparticacid-glutamic acid, and asparagine-glutamine.

As mentioned above, one example of an algorithm that is suitable fordetermining percent sequence identity and sequence similarity is theBLAST algorithm, which is described in Altschul et al., 1977, Nuc. AcidsRes. 25:3389-3402. BLAST is used, with the parameters described herein,to determine percent sequence identity for the nucleic acids andproteins of the present disclosure. Software for performing BLASTanalysis is publicly available through the National Center forBiotechnology Information (available at the following internet address:ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always>0)and N (penalty score for mismatching residues; always<0). For amino acidsequences, a scoring matrix is used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The term “toxin” refers to all peptides and/or proteins, of any aminoacid length and sequence, in either monomeric or multimeric formsnaturally, present in animal venoms or poisons and their non-venomhomologues. Animal toxins include all molecules identified or inferredby any means (e.g., physical, chemical, biochemical, genetic, genomic,proteomic) from animal venoms or poisons, including but not limited toisolation from crude venoms, isolation from venom gland tissues orextracts, identification based on venom gland proteome/proteomics,venome/venomics, transcriptome, and/or EST analysis. In someembodiments, a toxin is a toxin from a venom or poison of a snake,snail, scorpion, sea anemone, lizard, or a spider. Representativetoxins, and their amino acid sequences and source designations, arepresented in Tables and Figures herein (e.g., Table 1, Table 2, Table13, Example 20, FIG. 8, FIG. 9, FIG. 14, FIG. 15).

The term “toxin peptide”, as used herein, refers to polypeptides thathave structural and/or functional similarity to one or more toxins (andincludes such toxins). In some embodiments, a toxin peptide has an aminoacid sequence that is substantially identical to that of a toxin. Insome embodiments, a toxin peptide is less than 100, 90, 80, 70, 60, 50,40, 30, 20 or fewer amino acids long. In some embodiments, a toxinpeptide is more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acidslong. In some embodiments, a toxin peptide is between about 20 and about60 amino acids long. In some embodiments, a toxin peptide is betweenabout 30 and about 50 amino acids long. In some embodiments, a toxinpeptide has an amino acid sequence that includes a plurality ofcysteines. In some embodiments, such cysteines are located at positionscorresponding to positions 7 or 8, 13 or 14, 27 or 28, 32 or 33, and/or34 or 35 of a toxin. In some embodiments, toxin peptides have an aminoacid sequence that includes the following sequence:(X)_(m)C(X)_(n)C(X)_(n)C(X)_(o)C(X)_(n)CXC(X)_(m) (SEQ ID NO:______),wherein X is any amino acid, and wherein m=2-10 amino acids, n=3-5 aminoacids, and o=7-12 amino acids. In some embodiments, toxin peptides havean amino acid sequence that includes:(X)_(m)KC(X)_(n)QC(X)_(n)CK(X)_(o)KCM(X)_(n)CXC(X)_(m) (SEQ IDNO:______), wherein X is any amino acid, and wherein m=2-10 amino acids,n=3-4 amino acids, and o=7-12 amino acids. In some embodiments, toxinpeptides have an amino acid sequence that includes:XXXKCXXXXQCXXXCKXXXXXXXKCMXXXCXCXX (SEQ ID NO:______), wherein X is anyamino acid. In some embodiments, a toxin peptide has an amino acidsequence that includes a plurality of sequence elements, each of whichis found in a natural toxin. In some embodiments, a toxin peptide has anamino acid sequence that includes a plurality of sequence elements thatare found in (or share substantially identity with sequence elementsthat are found in) a plurality of different natural toxins. In someembodiments, a toxin peptide has an amino acid sequence that includes atleast two sequence elements that are found in (or share substantiallyidentity with sequence elements that are found in) the same naturaltoxin, but further includes one or more sequence elements that are notfound in the natural toxin.

The term “transmembrane protein” refers to polypeptides that partiallyspan a membrane and that completely span a membrane. A “transmembraneprotein” refers to monomeric as well as multimeric proteins, includingheteromultimeric proteins. A transmembrane protein can be a proteinfound on any membrane of a cell (e.g., a membrane of an intracellularcompartment such as the endoplasmic reticulum, Golgi apparatus, anendocytic compartment, nuclear membrane, or a cell surface membrane).

The term “wild-type”, when applied to a polypeptide (e.g., a receptorpolypeptide, or a toxin polypeptide) refers to a polypeptide whoseprimary amino acid sequence is identical to that of a polypeptide foundin nature. As will be appreciated by those skilled in the art, a wildtype polypeptide is one whose amino acid sequence is found in normal(i.e., non-mutant) polypeptides.

Receptors

Methods described herein are applicable to any receptor, i.e., anyreceptor that can serve as an interacting partner for a ligand. In mostembodiments, a receptor suitable for methods described herein is areceptor that serves as an interacting partner for a toxin peptide. Insome embodiments, a receptor is a transmembrane protein. In someembodiments, a receptor is a channel protein. Exemplary receptors fortoxin peptides may include, but are not limited to, ion channels (e.g.,potassium, sodium, calcium, chloride, and non-specific ion channels), aswell as other transmembrane proteins that are sensitive to toxinpeptides, for example, neurotransmitter receptors (e.g., NMDA receptor,serotonin (5-HT) receptor, alpha1-adrenoceptor, muscle-type nicotinicacetylcholine receptor (nAChR), neuronal-type nAChR, muscarinicacetylcholine (ACh) receptor), receptors for endogenous peptides (e.g.,neurotensin receptor, endothelin receptor, natriuretic peptide receptor,vasopressin receptor), noradrenaline transporter, acetylcholinesterase,GPIIb/IIIa integrin receptor, angiotensin-converting enzyme, andG-protein coupled receptors that are and are not ion channels. Receptorsalso include amino acid transporters and integrin receptors (e.g.,glycoprotein IIb/IIIa integrin receptors). Table 1 below provides anon-exclusive list of receptors, as well as exemplary toxin ligands forthe receptors, and organisms in which the ligands are naturallyexpressed. Receptors that may be utilized in accordance with the presentinvention include wild type receptors and also receptor polypeptideswhose amino acid sequences are substantially identical to wild typepolypeptides. Furthermore, as will be appreciated by those skilled inthe art, receptor polypeptides with various sequence modifications(e.g., fusions, substitutions, deletions, additions, rearrangements) ascompared with a wild type receptor may be utilized if desired.

TABLE 1 Organism Ligand (scaffold) name Representative receptor (target)marine Conus geographus GIIIA Na⁺ channel snail sea Stichodactylahelianthus Shk toxin K⁺ channel (e.g., Kv1.1) anemone scorpionkaliotoxin K⁺ channel (e.g., Kv1.3) scorpion hongotoxin-1 K⁺ channel(e.g., Kv1.1) scorpion Odontobuthus doriae OD 1 toxin Na⁺ channel spiderGrammostola spatulata voltage sensor toxin K⁺ channel (e.g., KvAP, KvAPVSD) (VSTX-1) spider Thrixopelma pruriens Protoxin-1 Na⁺ channel snakethree-finger toxins acetylcholine receptor snake Dendroaspis natriureticpeptide natriuretic peptide receptor A snake sarafotoxin endothelinreceptor (e.g., endothelin receptor B) snake dendrotoxin K⁺ channel(e.g., Kv1.1), Ca⁺ channel snake ADAM disintegrin/metalloproteinaseintegrins/extracellular matrix snake cobra venom factor complementsystem snake CNP-BPP snake CRISP snake crotamine snake cystatin snakefactor V snake factor X snake Fasciculin-2 Acetylcholinesterase snakekallikrein snake L-amino acid oxidase snake mamba intestinal toxin snakenerve growth factor snake phospholipase A2 type IB Phospholipids snakephospholipase A2 type IIA Phospholipids snake SPRY SPla/ryanodine snakeVEGF snake waglerin acetylcholine receptor snake waprin marineκ-conotoxins PVIIA K⁺ channel snail marine κA-conotoxins SVIA K⁺ channelsnail marine κM-conotoxins RIM( K⁺ channel snail marine μ-conotoxinPIIIA Na⁺ channel snail marine μO-Conotoxin MrVIB Na⁺ channel snailmarine δ-conotoxin TxVIA Na⁺ channel snail marine ziconotide/conotoxinsN Ca²⁺ channel snail marine ω-conotoxin CVID N Ca²⁺ channel snail marineω-conotoxin NVIID N Ca²⁺ channel snail marine ω-conotoxin MVIIC P/Q Ca²⁺channels snail marine α-conotoxin GI muscle nAChR snail marineαA-conotoxin PIVA muscle nAChR snail marine ψ-conotoxin PIIIE musclenAChR snail marine α-conotoxin Vc1.1 neuronal nAChR snail marineConantokin-G NMDA receptor snail marine Contulakin-G neurotensinreceptor snail sea Sea anemone Type 1 ShK K⁺ channel anemone sea Seaanemone Type 2 K⁺ channel anemone sea Sea anemone Type 3 K⁺ channelanemone sea 1 Sea anemone Type 1 ApB ApB Na⁺ channel anemone sea Seaanemone Type 2 Na⁺ channel anemone sea Sea anemone Type 1 + 2 Na⁺channel anemone sea Sea anemone Type 3 Na⁺ channel anemone sea Seaanemone Type Others Calitoxin I Na⁺ channel anemone sea APETx2 ASICchannels anemone venomous Helokinestatin bradykinin B2 receptor lizardsvenomous exendin-4 glucagon-like peptide 1 receptor lizards

According to methods of the present disclosure, a single ligand ormultiple ligands may be identified for one or more receptors. In someembodiments, receptors are expressed in cells. In some embodiments,receptors are immobilized (e.g., immobilized on a solid support or in anartificial membrane). In some embodiments, receptors are purified.

In some embodiments, a receptor is a potassium channel. Potassiumchannels are mainly found in plasma membranes but are not generallydistributed over the cell surface. Potassium channels catalyze the rapidpermeation of potassium ions while rejecting biologically abundantpotential competitors such as sodium, calcium and magnesium. Ionselectivity and high through put rate of potassium channels isaccomplished by precise co-ordination of dehydrated potassium by theprotein and multiple ion occupancy within the permeation pathway.

All potassium channels carry out the formation of a transmembrane “leak”specific for potassium ions. Since cells almost universally maintaincytoplasmic potassium concentrations higher than those extracellularly,the opening of a potassium channel implies a negative ongoing change inelectrical voltage across the cell membrane. This may result intermination of the action potential of electrically excitable cellsincluding nerve, muscle and pancreatic beta cells. In non-excitablecells, potassium channels play important roles in the cellular potassiumrecycling required for electrolyte balance affected by the renalepithelium.

In some embodiments, a receptor is a voltage gated potassium channelbelonging to the delayed rectifier class or Shaker potassium channelsubfamily, which includes Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6,Kv1.7, and Kv1.8. In some embodiments, a receptor is a potassiumreceptor of the Kv2, Kv3, Kv4, Kv5, Kv6, Kv7, Kv9, Kv9, Kv10, Kv11, orKv12 family (see an exemplary list of such receptors in Gutman et al.,Pharmacol. Rev. 57(4): 473-508, 2005).

In some embodiments, a receptor is a Kv1.3 potassium channel. Kv1.3potassium channels are a voltage-gated receptors expressed in a numberof tissues, including T lymphocytes. One exemplary a ligand for Kv1.3channel is kaliotoxin (KTX). KTX, found naturally in the venom of atleast one species of scorpions, is a peptidyl inhibitor ofCa(2+)-activated K⁺ channels and voltage-gated K⁺ channels Kv1.1, Kv1.2,Kv1.3. KTX is a single, approximately 4-kDa, polypeptide chain. KTXdisplays sequence homology with other scorpion-derived inhibitors ofCa(2+)-activated or voltage-gated K⁺ channels: 44% homology withcharybdotoxin (CTX), 52% with noxiustoxin (NTX), and 44% withiberiotoxin (IbTX).

In certain embodiments, a receptor is a voltage-gated sodium channel.Sodium channels have important functions throughout the body. Forexample, Nav1.4 controls excitability of skeletal muscle. Nav1.5controls excitability of cardiac myocytes. Nav1.1, Nav1.2, and Nav1.6are abundant in the central nervous system. Nav1.8 and Nav1.9 areexpressed in sensory neurons and have a role in pain perception. Nav1.7is broadly expressed in the peripheral nervous system and plays a rolein the regulation of action potential.

Ligands and Ligand Libraries

Among other things, the present disclosure provides methods foridentifying, characterizing, and/or detecting ligands for receptors. Invarious embodiments, ligands are toxin peptides (e.g., toxin peptidesderived from an animal venom). Provided methods can include the use oflibraries of toxin peptides to permit simultaneous screening of multiplecandidate ligand species. Methods herein are applicable to identifyingligands that derive from (i.e., are structurally related to) any toxin.For example, methods are applicable to toxin peptides which are derivedfrom toxins of organisms such as sea anemone (e.g., Stichodactylahelianthus), scorpion (e.g., Androctonus mauretanicus, Odontobuthusdoriae), snakes (e.g., Dendroaspis), spiders (e.g., tarantula), andsnails.

Toxins that can serve as a scaffold for toxin peptides and libraries oftoxin peptides include any of the toxins listed in a Table or Figureherein. For example, one or more of kaliotoxin, dendrotoxin, ShK toxin,hongotoxin-1, tarantula venom toxin vstx1, hanatoxin, fasciculin-2,dendroaspis natriuretic peptide (DNP), sarafotoxin, Odontobuthus doriaeOD1 toxin, Thrixopelma pruriens prototoxin-1, Thrixopelma pruriensprototoxin-2 can serve as a scaffold for generating a toxin peptide orlibrary toxin peptides.

Odontobuthus doriae OD1 toxin is a ligand for voltage gated sodiumchannels. OD1 toxin has the following amino acid sequence:

(SEQ ID NO: _(——)) GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR

Thrixopelma pruriens Protoxin-1 ProTx-I toxin is a toxin for voltagegated sodium channels. ProTx-I toxin has the following amino acidsequence:

(SEQ ID NO: _(——)) ECRYWLGGCSAGQTCCKHLVCSRRHGWCVWDGTFS.

Toxin peptides and toxin peptide libraries can be generated by any of avariety of methods. Variability of toxin peptide sequences can derivefrom combinatorial diversity and/or introduction of sequence variation.For example, in some embodiments, a toxin peptide has an amino acidsequence which includes one or more domains from one toxin, and one ormore domains from one or more other toxins. In one example, a toxinpeptide includes a first domain from a first toxin, a second domain froma second toxin, and a third domain from a third toxin. In someembodiments, a toxin peptide has an amino acid sequence of a naturaltoxin which has been altered such that the toxin peptide has an aminoacid sequence with at least 40%, 50%, 60%, 70%, 80%, 90%, 95% sequenceidentity to the natural toxin sequence. Sequence alterations suitablefor generation of toxin peptides include insertions, deletions,substitutions, rearrangements (e.g., inversions) and combinationsthereof. In some embodiments, sequence alterations are introduced into atoxin sequence at random. In some embodiments, sequence alterations areintroduced into a toxin sequence in a targeted manner (e.g., to varyresidues within a particular domain). In some embodiments, sequencealterations are introduced at residues other than cysteine residues(e.g., to preserve disulfide bonding). In some embodiments, sequencealterations are introduced at residues other than cysteine residues andbasic residues. In some embodiments, a toxin peptide has an amino acidsequence of a natural toxin which has been altered at residues thatundergo posttranslational modifications. Any and/or all of thesefeatures can be used to generate a diverse library of peptides forscreening and identifying novel ligands.

In some embodiments, multiple toxins serve as a scaffold source for asingle ligand, thereby providing combinatorial diversity to the range ofligand sequences that can be produced, screened, and utilized in amethod described herein. A group of toxins suitable for use as ascaffold for a ligand (i.e., a “scaffold group”) typically has a similarnumber of amino acids, and a similar arrangement of disulfide bridges.Members of a scaffold group typically have a homologousthree-dimensional backbone structure. Members of a scaffold group canhave very different biological properties and structural variation.

In some embodiments, members of a library include sequences that includecysteines spaced at intervals observed in a natural toxin, withvariability in residues between cysteines. In certain embodiments, oneor more additional residues found in a natural toxin sequence areconserved (e.g., basic residues known to be important for bindingactivity). In some embodiments, kaliotoxin-1 serves as a scaffold for aligand or library thereof. In certain embodiments, members of a libraryinclude peptides that have cysteines spaced at intervals observed inkaliotoxin, i.e., the peptides include the underlined cysteines found inkaliotoxin-1: GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK (SEQ ID NO:______).Such peptides have the following consensus sequence:XXXXCXXXXXCXXXCXXXXXXXXXCXXXXCXCXX (SEQ ID NO:______), wherein X is anyamino acid.

In certain embodiments, members of a library include peptides havingadditional residues conserved from the kaliotoxin-1 sequence, such asthe underlined residues: GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK (SEQ IDNO:______). Such peptides have the following consensus sequence:XXXXXXKCXXXXQCXXXCXXXXXXXXKCMXXKCXCXXX (SEQ ID NO:______) wherein X isany amino acid.

In certain embodiments, members of a library include peptides havingstill additional residues conserved from the kaliotoxin-1 sequence, suchas the underlined residues: GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPK (SEQID NO:______). Such peptides have the following consensus sequence:XXXXXXKCXXXXQCLXXCKXXXXXXXKCMXXKCXCXXX (SEQ ID NO:______) wherein X isany amino acid. Libraries can include peptides having residues conservedfrom other toxins, e.g., other toxins described herein, e.g., one ormore toxins from Table 1 or Table 2, Table 13, Example 20, FIG. 8, FIG.9, FIG. 14, or FIG. 15.

In certain embodiments, the present disclosure provides a ligand thatincludes the following consensus sequence:IXVKCXXPXQCXXPCKXXXGXXXXXKCMNXKCXCYX (SEQ ID NO: ______), wherein X isany amino acid. In certain embodiments, the present disclosure providesa ligand that includes the following consensus sequence:IXVKCXXPXQCXXPCKXXGXXXXKCMNXKCXCYX (SEQ ID NO: ______), wherein X is anyamino acid. In some embodiments, the ligands specifically bind to apotassium channel. In some embodiments, the ligands inhibit a potassiumchannel.

Table 2 provides an exemplary list of toxins that share a scaffold withkaliotoxin.

TABLE 2 An Exemplary Kaliotoxin Scaffold Group SEQ ID namescaffold member amino acid sequence NO: Kaliotoxin-1 (KTX)GVEINVKCSGSPQCLKPCKD--AGMRFGKCMNRKCHCTPK KTX_Androctonus_mauKTX2_Androctonus_aus -VRIPVSCKHSGQCLKPCKD--AGMRFGKCMNGKCDCTPK alpha--VGIPVSCKHSGQCIKPCKD--AGMRFGKCMNRKCDCTPK KTx_3_9_KAX39_BUTOC*alpha-KTx_3_6_AF079062_2 GVGINVKCKHSGQCLKPCKD--AGMRFGKCINGKCDCTPKGalpha-KTx_3_8KAX38_BUTSI GVPINVKCRGSPQCIQPCRD--AGMRFGKCMNGKCHCIPQ alpha-AVRIPVSCKHSGQCLKPCKD--AGMRFGKCMNGKCDCTPK KTx_3_5_KAX35_ANDAUBmKTX_Buthus_mar -VGINVKCKHSGQCLKPCKD--AGMRFGKCINGKCDCTPKAgTX1_Leiurus_qui GVPINVKCTGSPQCLKPCKD--AGMRFGKCINGKCHCTPKAgTX2_Leiurus_qui GVPINVSCTGSPQCIKPCKD--AGMRFGKCMNRKCHCTPKAgTX3_Leiurus_qui GVPINVPCTGSPQCIKPCKD--AGMREGKCMNRKCHCTPKOsK-1_Orthochirus_scr GVIINVKCKISRQCLEPCKK--AGMRFGKCMNGKCHCTPKNTX_Centruroides_nox TI-INVKCTSPKQCSKPCKELYGSSAGAKCMNGKCKCYNNKAX28_CENEL_alpha- TV-INVKCTSPKQCLKPCKDLYGPHAGAKCMNGKCKCYNNKTx_2_8_Toxin_Ce1 KAX29_CENEL_alpha-TI-INVKCTSPKQCLKPCKDLYGPHAGAKCMNGKCKCYNN KTx2_9_Toxin_Ce2*KAX2A_CENEL_alpha- IF-INVKCSLPQQCLRPCKDRFGQHAGGKCINGKCKCYP-KTx_2_10_Toxin_Ce3 *KAX2B_CENEL_alpha-TI-INVKCTSPKQCLLPCKEIYGIHAGAKCMNGKCKCYKI KTx_2_11_Toxin_Ce4*KAX2C_CENEL_alpha- TI-INVKCTSPKQCLPPCKEIYGRHAGAKCMNGKCHCSKIKTx_2_12_Toxin_Ce5 KAX23_CENLL_alpha-IT-INVKCTSPQQCLRPCKDRFGQHAGGKCINGKCKCYP- KTx_2_3_Toxin-1_C11Tx1AAB32772_1_toxin_1_(—) IT-INVKCTSPQQCLRPCKDRFGQHAGKGCINGKCKCYP-Centruroides_lim alpha-KTx_4_5_precursor_(—)VF-INVKCRGSPECLPKCKEAIGKSAG-KCMNGKCKCYP- Tityus_cosalpha-KTx_1_5_precursor_(—) QF-TDVKCTGSKQCWPVCKQMFGKPNG-KCMNGKCRCYS-Mesobuthus_mar TsTX-Kalpha_Tityus_serVF-INAKCRGSPECLPKCKEAIGKAAG-KCMNGKCKCYP- alpha-KTx_4_5_KAX45_TITCOVF-INVKCRGSPECLPKCKEAIGKSAG-KCMNGKCKCYP- MgTX_Centruroides_marTI-INVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH KAX22_CENMA_alpha-TI-INVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCY-- KTx_2_2_Margatoxin_MgTXNTX2_Centruroides_nox TI-INEKCFATSQCWTPCKKAIGSLQS-KCMNGKCKCYNGPiTX-Kalpha_Pandinus_imp TI-S---CTNPKQCYPHCKKETGYPN-AKCMNRKCKCFGRPiTX-Kbeta_Pandinus_imp TI-S---CTNEKQCYPHCKKETGYPN-AKCMNRKCKCFGRC1TX_Centruroides_lim IT-INVKCTSPQQCLRPCKDRFGQHAGGKCINGKCKCYP-KAX25_CENLM_alpha- TV-IDVKCTSPKQCLPPCKAQFGIRAGAKCMNGKCKCYPHKTx_2_5_Hongotoxin-1_HgTX1 ChTX_Leiurus_quiZF-TNVSCTTSKECWSVCQRLHNTSR-GKCMNKKCRCYS- IbTX_Buthus_tamZF-TDVDCSVSKECWSVCKDLFGVDR-GKCMGKKCRCYQ- Lq2_Leiurus_quiZF-TQESCTASNQCWSICKRLHNTNR-GKCMNKKCRCYS- Lq15-1_Leiurus_quiGL-IDVRCYDSRQCWIACKKVTGSTQ-GKCQNKQCRCY-- BmTX1_Buthus_marZF-TDVKCTGSKQCWPVCKQMFGKPN-GKCMNGKCRCYS- BmTX2_Buthus_marZF-TNVSCSASSQCWPVCKKLFGTYR-GKCMMSKCRCYS- LTX1_Leiurus_quiAF-----CNL-RMCQLSCRSL---GLLGKCIGDKCECVKH P05_Androctonus_mauTV-----CNL-RRCQLSCRSL---GLLGKCIGVKCECVKH BmP05_Buthus_marAV-----CNL-KRCQLSCRSL---GLLGKCIGDKCECVKH P01_Anroctonus_mau-----VSCE---DCPEHCSTQKAQ---AKCDNDKCVCEPI alpha-KTx 8.1 BmP01_Buthus_mar-----ATCE---DCPEHCATQNAR---AKCDNDKCVCEPK BmP02_Buthus_mar-----VGCE---ECPMHCKGKNAK---PTCDDGVCNCN-V BmP03_Buthus_mar-----VGCE---ECPMHCKGKNAN---PTCDDGVCNCN-V TsKappa_Tityus_serVV-IGQRCYRSPDCYSACKKLVGKAT-GKCTNGRCDC---

An alignment of peptides from exemplary scaffolds of toxins from Conusmarine snails are shown in FIG. 14, with disulfide bonding patternsindicated under each set of peptides (from French and Terlau, J. Med.Chem. 11:3053-3064, 2004). These Conus toxins target sodium channels.Another exemplary set of natural scaffolds that target sodium channelsare shown in FIG. 15 (from Mouhat et al., 378(Pt 3):717-26, 2004). Anyof these toxins can be used to produce toxin peptides. Libraries oftoxin peptides can be produced using methods described herein. In someembodiments, toxin peptides include cysteine at positions correspondingto cysteines found in the toxin(s). In some embodiments, toxin peptidesinclude one or more basic residues corresponding to basic residues foundin the toxin(s).

In one example, members of a scaffold group used to produce one or moreligands includes kaliotoxin-1, charybdotoxin, and agitotoxin-2. Thiscombination of toxins served as a source for the novel artificial ligandmokatoxin-1, as described in Examples herein. Mokatoxin-1 (also referredto as MK-1) is composed of A, B and C domains present in at least threedifferent species of scorpions, for example Buthus occitanus (AgTx2,domain A, North Africa), Centruroides elegans (Ce3, domain B, CentralAmerica), and Leiurus quinquestriatus (charybdotoxin, domain C, MiddleEast). As shown in FIG. 9, the sequence for mokatoxin-1 isINVKCSLPQQCIKPCKDAGMRFGKCMNKKCRCYS (SEQ ID NO: 1). Further novelneurotoxin-like sequences depicted in FIG. 9 include mokatoxin_(—)0422,TVIDVKCTSPKQCLPPCKAQFGIRAGAKCMNKKCRCYS (SEQ ID NO: 2), andmokatoxin_(—)0516, TVINVKCTSPKQCLRPCKDRFGQHAGGKCMNGKCKCYPH (SEQ ID NO:3).

Scaffolds are not limited to venom toxins. For example, certainnon-venom peptides from mammals share a scaffold with venom toxins. Suchpeptides can be employed in a scaffold group to produce novel ligands inaccordance with the present disclosure. To give one example, dendroaspisnatriuretic peptide from mamba snake venom and human brain natriureticpeptide fall into a scaffold group. Derivatives and/or portions of thesepeptides can be synthesized and/or combined to produce novel ligands.

Ligands (e.g., toxin peptides) can be produced by any method. In someembodiments, a ligand is produced by recombinant expression in a cell.In some embodiments, a ligand is produced by peptide synthesis. In someembodiments, a ligand is produced by in vitro translation.

Ligands can be inserted into vectors for expression and/or libraryselection. In some embodiments, a library is presented in a proteinarray (see, e.g., U.S. Pat. No. 5,143,854; De Wildt et al., Nat.Biotechnol. 18:989-994, 2000; Lueking et al., Anal. Biochem.270:103-111, 1999; Ge, Nucleic Acids Res. 28, e3, I-VII, 2000; MacBeathand Schreiber, Science 289:1760-1763, 2000; WO 01/98534, WO 01/83827, WO02/12893, WO 00/63701, WO 01/40803 and WO 99/51773). In someembodiments, a library is presented on a replicable genetic package,e.g., in the form of a phage library such as a phage display, yeastdisplay library, ribosome display, or nucleic acid-protein fusionlibrary. See, e.g., U.S. Pat. No. 5,223,409; Garrard et al. (1991)Bio/Technology 9:1373-1377; WO 03/029456; and U.S. Pat. No. 6,207,446.Binding members of such libraries can be obtained by selection andscreened in a high throughput format. See, e.g., U.S. 2003-0129659.

In one example, a nucleic acid sequence encoding a ligand may beinserted into a phagemid or phage vector, in-frame, to form aleader-linker-ligand-linker-coat protein construct (Clackson and Lowman,Phage display. Oxford University Press, 2004; Barbas et al., Phagedisplay. A laboratory manual. Cold Spring Harbor Laboratory Press,2001). For example, FIG. 1 schematically depicts KTX incorporated into aphagemid vector, in frame, fused by N and C-terminal linker sequencesbetween the leader sequence and the phage coat protein III. Exemplaryupstream and downstream leader amino acid sequences are AEGA (SEQ IDNO:______) and GSASSA (SEQ ID NO:______), respectively, and an exemplarycoat protein is protein III or its truncated version. Phages can begrown, prepared, titered and stored (Clackson and Lowman, Phage display.Oxford University Press, 2004; Barbas et al., Phage display. Alaboratory manual. Cold Spring Harbor Laboratory Press, 2001).

Ligand libraries for phage display can be generated by standard methods(Clackson and Lowman, Phage display. Oxford University Press, 2004;Barbas et al., Phage display. A laboratory manual. Cold Spring HarborLaboratory Press, 2001). In one example, ligand libraries for phagedisplay with a combinatorial arrangement of ligand-domains are generatedby designing overlapping or non-overlapping oligonucleotidescorresponding to each individual domain. These oligonucleotides arephosphorylated, annealed, mixed in a desired combination andconcentration and ligated into a phagemid vector with or without linkersequences to create a library by standard methods (Sambrook et al.,Molecular Cloning: A Laboratory Manual. Vols 1-3. Cold Spring HarborLaboratory Press, 1989). For example, ligands may be composed by domainsA1B1C1, A1B2C1, and A3B2C1, respectively. A combinatorial library ofligands in this representative example yields the pattern AnBnCn, wheren is the i-th domain (for example, A2B1C3 is a novel ligand present inthis library).

Library diversity is verified by sequencing or by other physical,chemical or biochemical means, either with or without statisticalanalysis, that is suitable to use for diversity verification. Domainscan be defined by functional, structural or sequence properties and canbe of any length and a domain can be present or absent. Domains can besingular or highly varied to expand diversity. Standard protocols ofmolecular biology may be employed (Sambrook et al., Molecular Cloning: ALaboratory Manual. Vols 1-3. Cold Spring Harbor Laboratory Press, 1989).

In one example, a library includes toxin peptides (e.g., toxin peptideshaving sequences from one or more animal toxins). Toxin peptidelibraries can include peptide animal toxins in their native or natural(wild-type) form or in any variation in amino acid sequence or may becomprised of DNA and/or RNA sequences encoding animal toxins. Thelibrary may contain toxins representing one or more scaffolds (alsoknown as toxin-types or toxin families). For example, in someembodiments, a library includes toxin peptides from scorpion toxins,venom three-finger molecular scaffolds, or animal toxins that interactwith K⁺ ion channels irrespective of the toxin's scaffold and speciesorigin. In some embodiments, a library includes all known toxins from agiven species, or all known toxins from all species.

In one example, an animal toxin library may include toxins from one ormore of sea anemone, scorpion, and snake. Sea anemone Stichodactylahelianthus ShK toxin is pharmacologically active and blocks Kv1.3 K⁺channels in mammalian cells when expressed on the phage. ScorpionAndroctonus mauretanicus kaliotoxin-1 is pharmacologically active andblocks Kv1.3 K⁺ channels in mammalian cells when expressed on the phage.Snake venom toxin Dendroaspis dendrotoxin is pharmacologically activeand blocks Kv1.1 K⁺ channels in mammalian cells when expressed on thephage (see FIG. 12, upper panel), while Camel VHH, a nonspecific-phage(see FIG. 12, middle panel) or phage buffer (see FIG. 12, bottom panel)cause no block.

In some embodiments, a library is incorporated into a phage displaysystem. In phage display, candidate ligands (e.g., toxin peptides) arefunctionally displayed on the surface of the phage and nucleic acidsequences encoding the ligands are enclosed inside phage particles. Thefunctional display permits the selection of ligands that interact with atarget or targets. A selection can be based on the ligand type (e.g.,toxin type) and/or target biochemistry, pharmacology, immunology and/orother physicochemical or biological property. For example, a K⁺ channeltoxin can be identified by screening scorpion toxin library on a K⁺channel for binding to the channel.

In one example, a toxin peptide library is constructed and maintainedsuch that each toxin peptide is individually constructed and stored andcan be mixed into the library in any desired combination for the test tobe performed. In another example, two or more toxin peptides may beconstructed in the same reaction and stored and used together.

A phage library can be transfected into E. coli or other suitablebacterial species, propagated and the phages purified. At this stage,ligands (e.g., toxin peptides) can be functionally expressed on thesurface of the phage and physically linked to their respective genesinside of the phage particle. A library is brought into contact with atarget. After incubation with the target, those phages that express aligand with no or weak recognition for the target are washed away. Theremaining ligands that interact with the target are dissociated and canbe (i) genotyped to establish the ligand identity, or (ii) processed forone or more rounds of panning, or (iii) otherwise quantified and/oridentified (e.g., ELISA, microbiological titering, functional testing).

A ligand library (e.g., toxin peptide library) may created by any knownmethod. For example, a toxin library may be created by collectingpeptides and/or nucleic acids encoding animal toxins and, if desired,non-venom homologues. Non-venom homologues include any molecule presentoutside of a venom gland or not used as a venom component but similar insequence or structure to toxins. In applications employing phagedisplay, N-terminal and C-terminal nucleotide sequences can be designedto join sequences for subcloning into a phagemid or other phage-displaycompatible vector. Overlapping or nonoverlapping DNA oligonucleotidesare designed and synthesized for synthetic genes. This includes positiveand negative DNA strands and N-terminal and C-terminal joining regions.The respective DNA oligonucleotide pairs (positive and negative strands)and sets are phosphorylated and annealed to create full length genes(e.g., genes encoding toxin peptides) with or without joining regions.Ligation into phagemid or other phage-display compatible vector isperformed, for example using coat protein III as a fusion protein. Othersuitable phage proteins may also be used. The sequences or genotypes canbe confirmed.

The strategy described above was used to produce a library of ligands.Amino acid sequences from members of the library are shown in FIG. 2.Seventy out of 120 (58.3%) sequences are scorpion K⁺ channel toxinscaffolds. MAAE (SEQ ID NO:______) is the C terminal part of the signalpeptide/secretion peptide-cleavage domain, position −4 to 0 relative tothe toxin sequence. GSASSA (SEQ ID NO:______) is an N-terminal linkerregion, which was placed immediately following the toxin sequence.

In some embodiments, a linker sequence used to connect a ligand (e.g.,toxin) sequence to a signal peptide and/or a coat protein of a phage(for phage display methods) and/or to any other domain is varied tooptimize one or more of ligand expression, binding, or function. Variedsequences can be produced by any method. Table 3 below lists linkersequences generated by Kunkel mutagenesis. “STM” corresponds to aC-terminal portion of one exemplary signal peptide. This is followed bya variable linker sequence of five amino acids, and an “AAK” sequence,which is one example of an N-terminal sequence that can follow a linkersegment.

TABLE 3 Exemplary Linker Segments for Expression  of Ligands SEQ ID STM..... AAK NO: 1 STM ADLHD AAK 2 STM ASTEF AAK 3 STM AVDGV AAK 4 STMCQPEL AAK 5 STM EQVDA AAK 6 STM GSDMH AAK 7 STM HTDYT AAK 8 STM LELTSAAK 9 STM LLTVP AAK 10 STM PLAGP AAK 11 STM SVSVS AAKMethods of Identifying, Characterizing, and/or Detecting Ligands

Ligands (e.g., toxin peptides) can be evaluated for binding to areceptor or for modulating activity of a receptor by any availablemethod.

Library Screening

The following provides exemplary methods for screening a displaylibrary. The methods can also be modified and used in combination withother types of libraries, e.g., an expression library or a proteinarray, and so forth. Ligands (e.g., a library of toxin peptides havingvaried amino acid sequences) can be displayed on phage, e.g.,filamentous phage. Library members having a desired degree of affinityfor and/or activity toward a receptor of interest can be identifiedusing immobilized or immobilizable receptors.

In some embodiments, a phage library is contacted with and allowed tobind to the target of interest. To facilitate separation of binders andnon-binders in the selection process, it is often convenient toimmobilize the receptor on a solid support, although it is also possibleto first permit binding to the target receptor in solution and thensegregate binders from non-binders by coupling the receptor to asupport. Bound phage may then be liberated from the receptor by a numberof means, such as changing the buffer to a relatively high acidic orbasic pH (e.g., pH 2 or pH 10), changing the ionic strength of thebuffer, adding denaturants, adding a competitor, adding host cells whichcan be infected (Hogan et al., Biotechniques 38(4):536, 538, 2005), orother known means.

In some embodiments, receptors are purified prior to ligand selection.For example, purified or partially purified natural, synthetic orsemi-synthetic receptors, such as KcsA carrying the toxin binding domainof a mammalian K⁺ channel Kv1.3, may be prepared and immobilized on asurface, such as a 96-well MaxiSorp (Nalgene Nunc International,Rochester, N.Y.) plate or comparable surface suitable for immobilizationand panning.

In another example, receptors may be expressed in cells. For example,cells may be stably or transiently transfected with one or morereceptors can be utilized as expressed in native tissues (Clackson andLowman, Phage display. Oxford University Press, 2004; Barbas et al.,Phage display. A laboratory manual. Cold Spring Harbor Laboratory Press,2001; Sambrook et al., Molecular Cloning: A Laboratory Manual. Vols 1-3.Cold Spring Harbor Laboratory Press, 1989). In one example, HEK(mammalian human embryonic kidney) and COS cells expressing wild-typeKv1.3 or Kv1.3-PDZ domain chimera were employed, the latter targetallowing a “double” panning (e.g., as a control or to enhance bindingaffinity) using phages expressing kaliotoxin-1 and/or PDZ-binder domain.

Panning may be performed by the binding of ligands to the receptors,followed by washes and ligand recovery. In one example, panning isperformed according to standard methods of phage display (Clackson andLowman, Phage display. Oxford University Press, 2004; Barbas et al.,Phage display. A laboratory manual. Cold Spring Harbor Laboratory Press,2001). Panning may be repeated until the desired enrichment is achieved.In addition, libraries can be pre-depleted on surfaces or cells thatcontain no receptors or on a receptor where the putative ligand receptordomain may be directly or indirectly altered. Additionally, any and allconditions of panning may be varied, altered or changed to achieveoptimal results, such as the isolation of a specific ligand. Panningvariations include, but are not limited to, the presence of competingligand(s), presence of excess target(s), length and temperature ofbinding, pre-absorption of the ligand library on one or more differentreceptor(s) or cells or surfaces, composition of binding solution (e.g.,ionic strength), stringency of washing, and recovery procedures. Phagesrecovered from panning may be processed for further rounds of panning,functional analysis, and/or sequencing/genotyping to deduce theresulting ligands' amino acid sequence or biological properties(Clackson and Lowman, Phage display. Oxford University Press, 2004;Barbas et al., Phage display. A laboratory manual. Cold Spring HarborLaboratory Press, 2001).

Following ligand recovery, ligands of interest may be produced in nativeform by standard methods of peptide/protein synthesis/production(Sambrook et al., Molecular Cloning: A Laboratory Manual. Vols 1-3. ColdSpring Harbor Laboratory Press, 1989; Albericio, Solid-Phase Synthesis:A Practical Guide. CRC, 2000; Howl, Peptide Synthesis and Applications.Humana Press, 2005).

Characterization of Binding Interactions

The binding properties of a ligand (e.g., a toxin peptide) can bereadily assessed using various assay formats. Techniques useful forevaluating binding of a ligand (e.g., a toxin peptide) to a receptor(e.g., a channel protein) include ELISA, surface plasmon resonance,Biomolecular Interaction Analysis, and the like. In some embodiments,binding interactions are analyzed using an ELISA assay. For example, theligand to be evaluated is contacted to a microtitre plate whose bottomsurface has been coated with the target receptor, e.g., a limitingamount of the receptor. The ligand is contacted to the plate. The plateis washed with buffer to remove non-specifically bound ligands. Then theamount of the ligand bound to the plate is determined by probing theplate with an antibody that recognizes the ligand. For example, theligand can include an epitope tag. The antibody can be linked to anenzyme such as alkaline phosphatase, which produces a colorimetricproduct when appropriate substrates are provided. In the case where adisplay library member includes the protein to be tested, the antibodycan recognize a region that is constant among all display librarymembers, e.g., for a phage display library member, a major phage coatprotein.

A binding interaction between a ligand and a particular receptor can beanalyzed using surface plasmon resonance (SPR). For example, before orafter sequencing of a display library member present in a sample, andoptionally verified, e.g., by ELISA, the displayed ligand can beproduced in quantity and assayed for binding the target using SPR. SPRor real-time Biomolecular Interaction Analysis (BIA) detects biospecificinteractions in real time, without labeling any of the interactants(e.g., BIAcore). Changes in the mass at the binding surface (indicativeof a binding event) of the BIA chip result in alterations of therefractive index of light near the surface (the optical phenomenon ofsurface plasmon resonance (SPR)). The changes in the refractivitygenerate a detectable signal, which are measured as an indication ofreal-time reactions between biological molecules. Information from SPRcan be used to provide an accurate and quantitative measure of theequilibrium dissociation constant (K_(D)), and kinetic parameters,including k_(on) and k_(off), for the binding of a ligand (e.g., a toxinpeptide) to a target receptor (e.g., an ion channel). Such data can beused to compare different ligands. Information from SPR can also be usedto develop structure-activity relationship (SAR). For example, if theligands are all mutated variants of a single parental toxin or a set ofknown toxins, variant amino acids at given positions can be identifiedthat correlate with particular binding parameters, e.g., high affinityand slow k_(off). Additional methods for measuring binding affinitiesinclude nuclear magnetic resonance (NMR), and binding titrations (e.g.,using fluorescence energy transfer). Other solution measures forstudying binding properties include fluorescence resonance energytransfer (FRET), NMR, X-ray crystallography, molecular modeling, andmeasuring bound vs. free molecules. Measurement of bound vs. freemolecules can be accomplished with a KinExA instrument from SapidyneInstruments Inc., Boise, Id.

Characterization of Biological Activity

In addition to, or instead of, receptor binding, biological activity ofligands can be characterized in methods provided by the presentdisclosure. Biological activities of ligands can be characterized by anyavailable means. In some embodiments, a ligand is a toxin peptide and areceptor is an ion channel. In these embodiments, methods for assessingchannel activity can be employed. Methods for assessing ion channelactivity include, for example, assays that measure voltage, current,membrane potential, and ion flux.

In some embodiments, ligands are tested for activity toward recombinantor naturally expressed functional ion channels. Samples that includefunctional channels (e.g., cells or artificial membranes) can be treatedwith a ligand and compared to control samples (e.g., samples without theligand), to examine the extent of modulation. Changes in ion flux may beassessed by determining changes in polarization (i.e., electricalpotential) of a cell or membrane expressing a channel. In someembodiments, a change in cellular polarization is by measuring changesin current (thereby measuring changes in polarization) withvoltage-clamp and patch-clamp techniques, e.g., the “cell-attached”mode, the “inside-out” mode, and the “whole cell” mode (see, e.g.,Ackerman et al., New Engl. J. Med. 336:1575-1595, 1997). Whole cellcurrents can be determined using standard methodology (see, e.g., Hamilet al., PFlugers. Archiv. 391:85, 1981). Other assays includeradiolabeled rubidium flux assays and fluorescence assays usingvoltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al., J.Membrane Biol. 88:67-75, 1988; Daniel et al., J. Pharmacol. Meth.25:185-193, 1991; Holevinsky et al., J. Membrane Biology 137:59-70,1994). In some embodiments, ligands to be tested are present in therange from 1 pM to 100 mM. Other methods for assessing a ligand'seffects on ion flux are described in the Examples herein. In someembodiments, the ability of a ligand to modulate (e.g., inhibit) anon-target receptor is tested, in addition to its ability to modulate atarget receptor.

Ligands can be tested to evaluate other types of biological effects,such as effects downstream of receptor activity. For example, Kv 1.3channels are expressed in T lymphocytes. Inhibitors of Kv1.3 channelssuppress T cell activation in vitro and delayed type hypersensitivity invivo, and have immunosupporessive activity in animal models ofautoimmunity (Beeton et al., 98:13942-13947, 2001; Koo et al., J.Immunol. 5120-5128, 1997). Accordingly, a candidate ligand for a Kv1.3receptor can be evaluated for the ability to suppress T cell activationin vitro and/or T cell dependent pathologies in vivo. Assays appropriatefor other ligand-receptor combinations would be apparent to one of skillin the art. Various exemplary effects of ligands that may be determinedusing intact cells or animals include transmitter release (e.g.,dopamine), hormone release (e.g., insulin), transcriptional changes,cell volume changes (e.g., in red blood cells), changes in cellmetabolism such as cell growth or pH changes, and changes inintracellular second messengers such as Ca²⁺.

Ligands can be selected for their potency and selectivity of modulationof a target receptor. In some embodiments, a ligand is assayed for itspotency toward a panel of receptors and an IC₅₀ value is determined foreach. A ligand that demonstrates a low IC₅₀ value for the targetreceptor, and a higher IC₅₀ value for other receptors within the testpanel, is considered to be selective toward the target receptor.Generally, a ligand is deemed selective if its IC₅₀ value is at leastone order of magnitude less than the next smallest IC₅₀ value measuredin the panel.

Pharmaceutical Compositions & Treatments

The present disclosure also features compositions including novelligands described herein. In some embodiments, a composition is apharmaceutically acceptable composition that includes a novel liganddescribed herein. The pharmaceutical composition can include apharmaceutically acceptable carrier.

Exemplary carriers include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Preferably, the carrier is suitable for oral, intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the ligand may be coated in a material to protect thecompound from the action of acids and other natural conditions that mayinactivate the compound.

The pharmaceutical composition can include a pharmaceutically acceptablesalt, e.g., a salt that retains the desired biological activity of theligand and does not impart any undesired toxicological effects (seee.g., Berge, S. M., et al., J. Pharm. Sci. 66:1-19, 1977).

The pharmaceutical composition may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes and suppositories. The preferred form dependson the intended mode of administration and therapeutic application.Typical compositions are in the form of injectable or infusiblesolutions, such as compositions similar to those used for administrationof antibodies to humans. A common mode of administration is parenteral(e.g., intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion). In one embodiment, the ligand composition is administered byintravenous infusion or injection. In another embodiment, the ligandcomposition is administered by intramuscular or subcutaneous injection.In another embodiment, the ligand composition is administered orally. Insome embodiments, the ligand composition is administered topically. Insome embodiments, the ligand composition is administered transdermally.Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage.

The composition including a ligand can be formulated as a solution,microemulsion, dispersion, liposome, or other ordered structure suitableto high drug concentration. Sterile injectable solutions can be preparedby incorporating the ligand in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying that yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The proper fluidityof a solution can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prolongedabsorption of injectable compositions can be brought about by includingin the composition an agent that delays absorption, for example,monostearate salts and gelatin.

Novel ligands described herein can be administered by a variety ofmethods known in the art. For many applications, the route/mode ofadministration is intravenous injection or infusion. For example, fortherapeutic applications, a ligand composition can be administered byintravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/minto reach a dose of about 1 to 100 mg/m² or 7 to 25 mg/m². Alternatively,the dose could be 100 μg/Kg, 500 μg/Kg, 1 mg/Kg, 5 mg/Kg, 10 mg/Kg, ormg/Kg. The route and/or mode of administration will vary depending uponthe desired results.

In certain embodiments, a ligand is prepared with a carrier thatprotects against rapid release, such as a controlled releaseformulation, including implants, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known. See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical formulation isa well-established art, and is further described in Gennaro (ed.),Remington: The Science and Practice of Pharmacy, 20.sup.th ed.,Lippincott, Williams & Wilkins, 2000 (ISBN: 0683306472); Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 7.sup.th Ed.,Lippincott Williams & Wilkins Publishers, 1999 (ISBN: 0683305727); andKibbe (ed.), Handbook of Pharmaceutical Excipients AmericanPharmaceutical Association, 3^(rd) ed., 2000 (ISBN: 091733096X).

Also provided by the present disclosure are kits that include a liganddescribed herein and instructions for use, e.g., treatment,prophylactic, or diagnostic use.

In addition to the ligand, the composition of the kit can include otheringredients, such as a solvent or buffer, a stabilizer or apreservative, and/or a second agent for treating a condition or disorderdescribed herein. Alternatively, the other ingredients can be includedin the kit, but in different compositions or containers than the ligand.In such embodiments, the kit can include instructions for admixing theligand and the other ingredients, or for using the ligand together withthe other ingredients.

A novel ligand described herein (e.g., a mokatoxin) can be administered,alone or in combination with, a second agent to a subject, e.g., apatient, e.g., a patient, who has a disorder (e.g., a Kv1.3-mediateddisorder), a symptom of a disorder or a predisposition toward adisorder, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disorder, the symptoms of thedisorder or the predisposition toward the disorder. The treatment mayalso delay onset, e.g., prevent onset, or prevent deterioration of acondition.

A therapeutically effective amount can be administered, typically anamount of the ligand which is effective, upon single or multiple doseadministration to a subject, in treating a subject, e.g., curing,alleviating, relieving or improving at least one symptom of a disorderin a subject to a degree beyond that expected in the absence of suchtreatment. A therapeutically effective amount of the composition mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the compound to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the compositionis outweighed by the therapeutically beneficial effects. Atherapeutically effective dosage preferably modulates a measurableparameter, favorably, relative to untreated subjects. The ability of aligand to inhibit a measurable parameter can be evaluated in an animalmodel system predictive of efficacy in a human disorder.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofligand calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a ligand described herein is 0.1-20mg/Kg, more preferably 1-10 mg/Kg. The compound can be administered byparenteral (e.g., intravenous or subcutaneous) infusion at a rate ofless than 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 50 mg/m²or about 5 to 20 mg/m². It is to be noted that dosage values may varywith the type and severity of the condition to be alleviated. It is tobe further understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions (e.g., the supervisingphysician), and that dosage ranges set forth herein are only exemplary.

Therapeutic Uses

Novel ligands described herein may be used in therapies for thetreatment or prophylaxis of disorders mediated target receptors (e.g.,Kv1.3 channels). Kv1.3 channels are a therapeutic target forimmunosuppressants. Accordingly, novel ligands described herein thatinhibit activity of Kv1.3 channels are useful in the treatment of immunedisorders. In some embodiments, a novel ligand is used to treatautoimmune and/or chronic inflammatory diseases, such as systemic lupuserythematosis, chronic rheumatoid arthritis, type I and II diabetesmellitus, inflammatory bowel disease, biliary cirrhosis, uveitis,multiple sclerosis, graft versus host disease, graft rejection, andother disorders such as Crohn's disease, ulcerative colitis, bullouspemphigoid, sarcoidosis, psoriasis, ichthyosis, Sjogren's syndrome,scleroderma, mixed connective tissue disease, dermatomyositis,polymyositis, Reiter's syndrome, Behcet's disease, myasthenia gravis,encephalomyelitis, Graves opthalmopathy, psoriasis, neurodermitis andasthma.

While various aspects and examples have been described, it will beapparent to those of ordinary skill in the art that many more examplesand implementations are possible within the scope of the invention.Accordingly, the disclosure is not to be restricted except in light ofthe attached claims and their equivalents.

EXAMPLES

Reference will now be made in detail to several examples. While thedisclosure will be described in conjunction with these examples, it willbe understood that it is not intended to limit the claimed invention tosuch examples. In the following description, numerous specific detailsare set forth in the examples in order to provide a thoroughunderstanding of the subject matter of the claims which, however, may bepracticed without some or all of these specific details. In otherinstances, well known process operations have not been described indetail in order not to unnecessarily obscure the description.

Example 1 Live Cells—Kv1.3 Cells vs. Vector

In one example, KTX was preferentially bound to HEK and COS cellsexpressing Kv1.3 channels vs. cells not expressing Kv1.3 (vector). Asshown in FIG. 3, panning on Kv1.3 transfected cells compared to thevector cells enriches for KTX. “A” shows about a 10-fold enrichment ofKTX when screened on COS cells transfected with Kv1.3 vs. the vector.5×10¹¹ phagemids were added to the cell cultures in “A.” “B” shows anapproximate 20-fold enrichment of KTX when screened on HEK cellstransfected with Kv1.3 vs. the vector. 5×10¹¹ phagemids were added tothe cell cultures in “B.”

Example 2 Live Cells—KTX Ligands vs. Non-Specific Ligands

In another example, KTX was preferentially enriched when a mixture ofnon-specific and KTX ligands was panned on cells expressing Kv1.3channels. As shown in Table 4, KTX was selected over non-KTX ligandswhen panning was performed on mammalian cells expressing Kv1.3 channels.

TABLE 4 INPUT OUTPUT KTX phages/nonspecific phages (%) Fold CalculatedActual KTX (%) Enrichment 10/90  7/93 (n = 30) 30 (n = 30) 4.3 1/99 5/95(n = 20) 26 (n = 20) 5.2 10/90  0/100 (n = 10)  22 (n = 18) enrichment

In the first input, the calculated ratio was 10 KTX phagemids per 100phages. The actual ratio, averaged over 30 samples, was 7 KTX phagemidsper 100 phages. After one round of panning, the output ratio increasedto 30 KTX phagemids per 100 phages, resulting in a 4.3 fold enrichment.

In the second input, the calculated ratio was 1 KTX phagemid per 100phages. The actual ratio, averaged over 20 samples, was 5 KTX phagemidsper 100 phages. After one round of panning, the output ratio increasedto 26 KTX phagemids per 100 phages, resulting in a 5.2 fold enrichment.

In the third input, the calculated ratio was 10 KTX phagemid per 100phages. The actual ratio, averaged over 10 samples, was 0 KTX phagemidsper 100 phages. However, after one round of panning, the output ratioincreased to 22 KTX phagemids per 100 phages. The third input showsenrichment of KTX, however the enrichment cannot be quantified due to noKTX phages being present in the 10 input samples.

Example 3 Live Cells—KTX-Like Ligands vs. Non-KTX-Like Ligands

In a further example, KTX-like ligands (e.g., having a KTX-like proteinscaffold) were preferentially enriched when a mixture of non-specificand KTX-like ligands were panned on cells expressing Kv1.3 channels. Alibrary of KTX-like ligands, with a calculated diversity of greater than11,000, was created de novo. The library comprised approximately 8%KTX-like ligands and 92% non-KTX-like ligands. The library of ligandswas screened on Kv1.3 receptors expressed in HEK and COS cells. As shownin Table 5, one round of panning (n=20-40 samples) specifically enrichedthe KTX-like ligands compared to the input library.

TABLE 5 Input Output Cell Type (% KTX-like ligands) (1 round of panning)HEK 8 40% KTX-like COS 8 35% KTX-like

Verification of the above Examples 1-3 was performed by quantificationof the elutes, and/or by genotyping the input versus output ligands.

Example 4 Purified Receptors—Enriching KTX from Mixture of Ligands

A mixture of five ligands: (1) kaliotoxin-1; (2) sarafotoxin s6b; (3)Dendroaspis natriuretic peptide (DNP); (4) fasciculin-2; and (5) camelantibody (CVHH); were mixed and panned on purified receptors(KcsA-Kv1.3). The control was immobilized antiserum to sarafotoxin s6b(anti-S6b). Input and output ligands were verified by genotyping. Inthis system, shown in Table 6, a 3.6-fold enrichment is seen afterone-round of panning.

TABLE 6 Output (% Ligands) Input (% Ligands) KcsA- Actual Kv.13 Anti-S6bLigand Calculated (n = 14) (n = 20) (n = 20) Kaliotoxin-1 20 28 100   0Sarafotoxin 20 36 0 100   s6b DNP 20 28 0 0 Fasciculin-2 20  0 0 0 CVHH20  7 0 0

Example 5 Purified Receptors—Enriching for Novel KTX-Like Ligands fromLibrary

A library of KTX-like ligands, with a calculated diversity of greaterthan 11,000, was created de novo. The library comprised approximately 8%KTX-like ligands and 92% non-KTX-like ligands. This library of ligandswas screened for novel ligands on (1) an immobilized target comprisingKv1.3 and (2) a control comprising KcsA. As shown in Table 7, a firstand second round of panning specifically enriches KTX-like ligands inKv1.3, but not in KcsA.

TABLE 7 % of KTX-like ligands (n = 20-40) Receptor Input 1^(st) Panning2^(nd) Panning Kv1.3 8 15  38  KcsA 8 0 0

Example 6 Purified Receptors—Quantification of Phages by ELISA

Panning was performed, in triplicate, on surfaces without a coating(none), and on immobilized KcsA-Kv1.3 receptors (KcsA-Kv1.3), wild-typeKcsA receptors (KcsA-WT), and antiserum to sarafotoxin S6b (anti-S6b).The input ligands per well are 10⁹, 10⁸, 10⁷. The ligands included:sarafotoxin S6b (upper panel FIG. 4), kaliotoxin-1 (KTX, middle row FIG.4), no ligand helper phages (bottom row FIG. 4). The data, shown in FIG.4, indicates specific binding of sarafotoxin S6b to anti-S6b, and KTX toKcsA-Kv1.3.

Example 7 DNP and Saratoxin S6b—Antiserum Reacts with Phages

As depicted in FIG. 5, Dendroaspis natriuretic peptide (DNP) andsarafotoxin S6b (S6b) ligands in a phage display system arepreferentially selected on antiserum raised against DNP and S6b,respectively. Based on its structural similarity, S6b also selected, butin a lesser extent, on antiserum raised against endothelin. The controlsincluded DX88 (a kallikrein ligand) and CVHH (a camel-Ag ligand). Thenumber 8 or 9 following the ligand name, in FIG. 5, indicates 10⁸ or 10⁹phages per reaction, respectively.

Example 8 Mokatoxin-1, a Kv1.3-Specific Artificial Neurotoxin

To confirm that a natural animal venom neurotoxin can bind to a known Kvchannel site when expressed on the surface of a phage particle, aphagemid was constructed with KTX of the scorpion Androctonusmauretanicus encoded on the N-terminus and in-frame with phage coatprotein III. As a control, a phage expressing a mutant toxin, DDD-KTX,was synthesized. DDD-KTX does not bind to KTX sites because three basicresidues on the KTX interaction surface are altered to aspartate (R24D,K27D, R31D).

For each binding determination, 3 wells in a NUNC-Immuno MaxiSorp96-Well plates were coated overnight at 4° C. with 1 ug of KcsA-1.3 orKcsA in 50 ul of 100 mM NaHCO₃, 1 mM DDM, pH 9, then washed once withTris-HCl 50 mM, NaCl 150 mM pH7.5 containing 0.1% Tween 20, 1 mM DDM(TBST). Wells were then blocked at room temperature with 200 ul of Tris50 mM NaCl 150 mM pH 7.5, 1 mM DDM, (TBS) containing 0.5% BSA, thenwashed once with TBST. For each well, 108-1010 phages were added in 50ul of TBS containing 0.5% BSA and incubated on a rotary shaker at roomtemperature for 2 hrs. Following five washes with TBST, 50 ul ofanti-phage antibody-peroxidase conjugated TBST with 0.5% BSA was addedand incubated on a rotary shaker for 2 hours at room temperature, thenwashed 5 times with TBST and twice with TBS. Fifty (50) ul of 1 stepturbo-TMB-ELISA was added and the reaction is stopped by 50 μl of 2MH₂SO₄ and the absorbency was read at 450 nM.

As shown in the ELISA assay of FIG. 6, KTX is able to express, fold andbind adequately when exposed on the phage surface as a fusion partnerwith protein III. KTX-phages bind to a purified potassium channel (KcsAcarrying a segment of the Kv1.3 pore loop, KcsA-1.3) whereas KTX-phagedid not bind to wild KcsA nor did DDD-KTX-phage bind to either KcsA-1.3or KcsA. The data represented in FIG. 6 is the mean and S.E. recordedfrom 3 wells. “A” shows Kaliotoxin-1 phage (fKTX) binds to KcsA-Kv1.3but not to wild-type KcsA. “B” shows Inactive kaliotoxin-1 (R24D, K27D,R31D; fDDD-KTX) does not bind to KcsA-1.3.

To demonstrate KTX-phages inhibit Kv1.3 channels, Kv1.3 channelsexpressed in human embryonic kidney cells (HEK293) were studied usingwhole-cell patch-clamp. Phagemids were applied at 1 nM and then washedout. Half recovery was achieved at ˜3 min wash time. Plasmids weretransfected into cells with Lipofectamine 2000 (Invitrogen) according tothe manufacturer's instructions. Experiments were performed at 24 hr.Whole-cell patch-clamp was performed using an Axopatch 200B amplifierand pCLAMP software (Molecular Devices, Union City, Calif.) at filterand sampling frequencies of 5 and 25 kHz respectively. Kv1.3 currentswere evoked by 250 ms test pulses to 50 mV from −80 mV with a 5 secondinterpulse interval and studied in a bath solution comprising in mM: 1.3CaCl₂, 0.5 MgCl₂, 0.4 MgSO₄, 3.56 KCl, 0.44 KH₂PO₄, 139.7 NaCl, 0.34Na₂HPO₄, 5.5 glucose, 10 HEPES adjusted to pH 7.4 with NaOH. Electrodeswere fabricated from borosilicate glass (Clark, Kent, UK) and had aresistance of ˜5 MΩ when filled with a solution containing in mM: 136KCl, 1 MgCl₂, 2 K₂ATP, 5 EGTA, 10 HEPES adjusted to pH 7.2 with KOH.Electrodes were coated with Sigmacote (Sigma) prior to use.

As shown in FIG. 7, KTX-phage inhibits Kv1.3 channels. Application of 1nM KTX-phage blocked currents by ˜35% and inhibition was reversed byhalf in ˜20 minutes on washing the cells with buffer without phageparticles. Conversely, DDD-KTX-phage, phage expressing camel CVHHantibody fragments to an unrelated antigen or buffer used for the phagepreparation did not suppress or augment channel currents.

A phage-display library of the novel toxin scaffolds was designed basedon the KTX family of scaffolds (FIG. 8). Thirty-six (36) known KTXfamily toxin sequences were aligned using the six conserved cysteineresidues they employ to form three disulfide bonds. The sequences werethereby considered in three linear domains homologous to KTX residuesG1-P12 (domain A), L15-G26 (domain B) and N30 to K38 (domain C). Toxinswere then constructed from one linkage of one of 30 unique A domains, 22unique B domains and 17 unique C domains present in the 32 parenttoxins. Forward and reverse primers were synthesized for each 69 uniquedomains, phosphorylated and annealed. Ninety separate reactions wereused to anneal domains (in each reaction was 1 A domain, 7 or 8 Bdomains, and 17 C domains in equimolar proportions). These were ligatedinto pAS62 phagemids and transfected into XL1 E. coli. Joining sequencesbetween AB and BC were shared sites (in KTX these are Q13C14 andK₂₇T28M29) leading to a calculated library diversity of 11,220 toxinvariants including the 19 known toxins. Random genotyping of isolatesconfirmed expression of all domains and 68% toxin-bearing phage.

The library was applied to KcsA-1.3 immobilized onto a solid-phase.Following two rounds of panning, 16.6% of the eluted phage was a singlenovel neurotoxin-like sequence composed of A, B and C domains from threedifferent species of scorpions, shown in FIG. 9: Buthus occitanus(AgTx2, domain A, North Africa), Centruroides elegans (Ce3, domain B,Central America), and Leiurus quinquestriatus (charybdotoxin, domain C,Middle East). The new toxin was named mokatoxin-1 (MK-1) and was notobserved in control selective trials with KcsA.

To confirm that isolation of MK-1 was specific, a 1:15,000 mixture ofmokatoxin-1-phage and DDD-KTX-phage was applied to immobilized KcsA-1.3.Sixty percent of the phage isolated after two rounds of selection wasMK-1 while all isolates recovered after two rounds on the KcsA controlwere DDD-KTX.

MK-1-phage particles were confirmed to bind specifically to KcsA-1.3 andalso block Kv1.3 currents in mammalian cells. Thereafter, MK-1 wassynthesized. As depicted in FIG. 10, application of the MK-1 towild-type Kv1.3 channels expressed in Xenopus oocytes showedhalf-maximal blockade at 3 nM. The blocking of Kv1.3 by MK-1 isrepresented by filled circles; the blocking of Kv1.2 by MK-1 isrepresented by filled diamonds; and the blocking of Kv1.1 by MK-1 isrepresented by filled triangles.

Notably, as shown in Table 8, the pharmacological profile of mokatoxin-1(MK-1) was different than all three of its parent toxins and KTX.Whereas MK-1 blocks only Kv1.3 potently, KTX blocks Kv1.1, Kv1.2, Kv1.3and BK, AgTx2 blocks Kv1.1 and Kv1.3, Ce3 does not block Kv1.3 and CTXblocks Kv1.2, Kv1.3 and BK.

The demonstrated selectivity of MK-1 on Kv1.3 in respect of other K+channels is one significant example of the power and utilizationpotential of the present method describing the creation of toxinlibraries and their screening. Pharmacological selectivity (specificity)is one example of useful modifications (e.g., improving on existingtoxins or toxin scaffolds) such residue alterations to improve targetspecificity, affinity, impact on receptor function, to attach cargo fordelivery to specific cellular and molecular locations and/or similarnew/useful properties.

TABLE 8 Channel approximate IC50 (nM) Toxin Kv1.1 Kv1.2 Kv1.3 BKMK-1 >1000 ~680 ~3 >1000 KTX-1 0.1 1.4 0.5 ~5 AgTX2 0.04 26.8 0.004~1150 Ce3 ND ND ~366 ND CTX 1500 5.6 2.5 3

Example 9 Kaliotoxin-1-Phage Specifically Binds to and Enriched onAnimal Cells Expressing Functional Mammalian Wild-Type K+ Channel Kv1.3

HEK cells were transfected with an expression vector carrying K⁺ channelKv1.3 (TEST) or with empty vector that does not code for anytransmembrane protein (CONTROL). Transfection efficiency was monitoredwith cotransfection of gene encoding for the green fluorescence protein(GFP) and observing the fluorescence after 1-2 days followingtransfection. Under these conditions, functional expression of the Kv1.3channel was verified by electrophysiological, biochemical, andimmunological means.

At the peak expression time for Kv1.3 channels (1-2 days followingtransfection), cells were detached from the monolayer, washed andbrought into contact with a calculated mixture of 2% KTX-phage:2%mokatoxin-1-phage:96% inactive kaliotoxin-1 (DDD-KTX)-phage. KTX andmokatoxin-1 are Kv1.3 specific scorpion toxins. DDD-KTX is a mutantvariant of KTX that does not bind to or inhibit Kv1.3 channels.Following incubation of the cells with the phages, cells were subjectedto wash to remove the unbound/weakly bound phages. The remaining boundphages were eluted from the cells and transformed into XL1 E. coli, andplated onto LB plates with antibiotic selecting for the phagemids. After1 day, 16-20 colonies of transformed XL1 were randomly selected andgenotyped to establish the relative ratio (i.e., its departure from thecalculated initial input of 2:2:96%) of the three different phagespecies, before and after the 1st and 2nd rounds of panning.

Kv1.3 specific KTX-phage was enriched from a calculated initial input of2% to 40% (n=20) after the 2nd panning on Kv1.3 expressing cells, butnot on vector transfected cells. In addition, Kv1.3 specificmokatoxin-1-phage was enriched from a calculated initial input of 2%(n=20) to 15% (n=20) after the 2nd panning on Kv1.3 expressing cells,but not on vector transfected cells. DDD-KTX-phage proportion decreasedin the Kv1.3 expressing cells, while in the vector transfected cells,after the 2nd panning, this was the only species recovered in our sample(n=16).

Example 10 Isolation of Mokatoxin_(—)0422, a Novel K⁺ Channel Toxin,from a Toxin Phage-Display Library by Panning on Kv1.3 K+ ChannelsExpressed in Mammalian Cells

HEK cells were transfected with an expression vector carrying K⁺ channelKv1.3 (TEST) or with empty vector that does not code for anytransmembrane protein (CONTROL). Transfection efficiency was monitoredby observing fluorescence after 1-2 days following cotransfection of agene encoding for the green fluorescence protein (GFP). Under theseconditions, functional expression of the Kv1.3 channel was verified byelectrophysiological, biochemical, and immunological means.

At the peak expression time for Kv1.3 channels (1-2 days followingtransfection), cells were detached from the monolayer, washed andbrought into contact with ˜3×10¹¹ phages of a KTX scaffold combinatoriallibrary. The KTX scaffold combinatorial library construction comprises aphage-display library of novel toxin scaffolds designed based on the KTXfamily of scaffolds. Thirty-six known KTX family toxin sequences werealigned using the six conserved cysteine residues employed by KTX toform three disulfide bonds. The sequences were thereby considered inthree linear domains homologous to KTX residues G1-P12 (domain A),L15-G26 (domain B) and N30 to K38 (domain C). Toxins were thenconstructed from one linkage of one of 30 unique A domains, 22 unique Bdomains and 17 unique C domains present in the 32 parent toxins. Joiningsequences between AB and BC were shared sites (in KTX these are Q13C14and K27T28M29) leading to a calculated library diversity of 11,220 toxinvariants including the 19 known toxins. One such toxin ismokatoxin_(—)0422, whose amino acid sequenceTVIDVKCTSPKQCLPPCKAQFGIRAGAKCMNKKCRCYS (SEQ ID NO: 2) is depicted inFIG. 9. Random genotyping of isolates confirmed expression of alldomains and 58.3% (n=120) toxin-bearing phage.

Following incubation of the cells with the phages, cells were subjectedto wash to remove the unbound/weakly bound phages. The remaining boundphages were eluted from the cells and transformed into XL1 E. coli.Transformed XL1 was (1) amplified overnight in liquid media in thepresence of a helper phage and antibiotic to prepare a phage preparation(to used in subsequent rounds of panning), and (2) an aliquot of thetransformed XL1 was plated onto LB plates with antibiotic selecting forthe phagemid. The following day, 18-20 colonies of the plated XL1 wererandomly selected and genotyped to establish enrichment on Kv1.3 cellsversus control cells not expressing Kv1.3.

Electrophysiological recordings were performed with mokatoxin_(—)0422 toconfirm blocking of Kv1.3 channels. Phagemids were applied at 1 nMmokatoxin_(—)0422-displaying phage concentration and then washed out.Half recovery was achieved at ˜3 min wash time. Plasmids weretransfected into cells with Lipofectamine 2000 (Invitrogen) according tothe manufacturer's instructions. Experiments were performed at 24 hrs.Whole-cell patch-clamp was performed using an Axopatch 200B amplifierand pCLAMP software (Molecular Devices, Union City, Calif.) at filterand sampling frequencies of 5 and 25 kHz respectively. Kv1.3 currentswere evoked by 250 ms test pulses to 50 mV from −80 mV with a 5 secondinterpulse interval and studied in a bath solution comprising, in mM:1.3 CaCl₂, 0.5 MgCl₂, 0.4 MgSO₄, 3.56 KCl, 0.44 KH₂PO₄, 139.7 NaCl, 0.34Na₂HPO₄, 5.5 glucose, 10 HEPES adjusted to pH 7.4 with NaOH. Electrodeswere fabricated from borosilicate glass (Clark, Kent, UK) and had aresistance of ˜5 MΩ when filled with a solution containing in mM: 136KCl, 1 MgCl₂, 2 K₂ATP, 5 EGTA, 10 HEPES adjusted to pH 7.2 with KOH.Electrodes were coated with Sigmacote (Sigma) prior to use.

Following the 1st round of panning, no toxin showed signs of enrichment(n=20). Following the 2nd round of panning, mokatoxin_(—)0422 appearedat 11% (n=18), while all other toxins appeared only once in the sample(i.e., no indication of enrichment). Electrophysiological recording at0.1 nM mokatoxin_(—)0422-displaying phage concentration, shown in FIG.12, confirmed that mokatoxin_(—)0422 blocks Kv1.3 channels expressed inmammalian cells. The control, DDD-kaliotoxin-1 phage (kaliotoxin-1 inwhich the binding sited are eliminated), did not inhibit Kv1.3 currentin this system.

Example 11 Verification that Mokatoxin_(—)0422 is Specifically Enrichedon Cells Expressing Kv1.3 Channels

HEK cells were transfected with an expression vector carrying K⁺ channelKv1.3 (TEST) or with empty vector that does not code for anytransmembrane protein (CONTROL Transfection efficiency was monitored byobserving fluorescence after 1-2 days following cotransfection of a geneencoding for the green fluorescence protein (GFP). Under theseconditions, functional expression of the Kv1.3 channel was verified byelectrophysiological, biochemical, and immunological means. At the peakexpression time for Kv1.3 channels (1-2 days following transfection),cells were detached from the monolayer, washed and brought into contactwith a calculated mixture of 5% mokatoxin_(—)0422-phage:95% inactivekaliotoxin-1 (DDD-KTX)-phage.

As described above, mokatoxin_(—)0422 is a Kv1.3 specific scorpion toxinisolated from a scorpion-toxin phage display library, while DDD-KTX is amutant variant of kaliotoxin-1 that does not bind to or inhibit Kv1.3channels. Following incubation of the cells with the phages, cells weresubjected to wash to remove the unbound/weakly bound phages. Theremaining bound phages were eluted from the cells and transformed intoXL1 E. coli, and plated onto LB plates with antibiotic selecting for thephagemids. The following day, 19-20 colonies of transformed XL1 wererandomly selected and genotyped to establish the relative ratio (i.e.,its departure from the calculated initial input of 5:95%) of the twodifferent phage species, before and after the panning.

As shown in Table 9, Kv 1.3 specific mokatoxin_(—)0422-phage enrichedfrom a calculated initial input of 5% (measured input ofmokatoxin_(—)0422-phage was 0% in a n=20 sample) to a measured 90%(n=20) after the one round of panning on Kv1.3 expressing cells, but noton vector transfected cells.

TABLE 9 # of genotype ( %) Calculated (%) Input Test Controlmokatoxin_0422  5 0 (0)  18 (90) 1 (5) DDD-KTX  95 20 (100)  2 (10) 18(95) Total    100% 20 (100)  20 (100)  19 (100)

Example 12 Functional Expression of Sea Anemone Animal Toxin Scaffold onthe Phage and Specific Selection

Sea anemone K⁺ channel toxin (ShK) was expressed in a phage display. Asshown in Table 10, Shk phage is enriched on a target KcsA-1.3 but not inKcsA. The target KcsA-1.3 contains a receptor domain for Shk from K⁺channel Kv1.3.

TABLE 10 % phages (n = 17-20) INPUT OUTPUT phage calculated actualKcsA-1.3 KcsA Shk 1 0 20  0 Camel VHH 99  100  0 80 

As shown in FIG. 13, Shk expressed on the phage (Shk) blocks Kv1.3current in mammalian cells, confirming that the animal toxin isfunctionally expressed. Control phage (Ctrl) expressing a nonspecificligand has no effect on the current.

Example 13 Mokatoxin-1 is Selective Among Potassium Channels

Mokatoxin-1 was selected for its high affinity for Kv1.3. This toxinpeptide has a large difference in affinity for other channels that areimportant targets for the kaliotoxin scaffold. Mokatoxin-1 is unique ascompared to known natural toxins and their natural or point mutanthomologs.

To examine the effect of mokatoxin-1 on activity of potassium ionchannels, cDNA encoding Rattus Kv1.1 (NCBI accession numberNM_(—)173095), Rattus Kv1.2 (NM_(—)012970), Homo Kv1.3 (NM_(—)002232),or Mus BK (NM_(—)010610) were subcloned into oocyte expression vectorsbased on PCR3.1 and pGEM. cRNA synthesis was performed with T7polymerase and the mMessageMachine Kit (Ambion, Austin, Tex.) accordingto manufacturers instructions. cRNA concentrations were determinedspectroscopically. For two electrode voltage clamp recordings oocyteswere extracted from Xenopus laevis and defoliculated with CollagenaseType 2 (Worthington, Lakewood, N.J.). Oocytes were maintained in ND91solution containing 2 mM KCl, 91 mM NaCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and5 mM HEPES, pH 7.5, supplemented with 1% Penicillin/Streptomycin(Cellgro, Hemdon, Va.) and 0.1% Gentamycin sulfate (Gibco, Grand Island,N.Y.)) at 16° C. Stage V and VI oocytes were injected with 8 pg to 4 ngcRNA, and currents recorded 1 to 4 days later.

For dose-response measurements of toxin block of Kv1.1, Kv1.2, or Kv1.3peak currents were recorded during a 500 ms step to 0 mV from a holdingvoltage of −100 mV, followed by a 200 ms step to −135 mV every 30 s. Todetermine the kinetics of toxin block oocytes were held at −100 mV andstepped to the test voltage of 0 mV for 100 ms followed by a 200 msduration step to −135 mV every 2 s. BK currents were recorded during a50 ms step to +60 mV from a holding voltage of −80 mV, followed by a 40ms step to −100 mV every 3 s. The perfusion solution contained 2 mM KCl,96 mM NaCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5 mM HEPES, pH 7.5 and 0.1% fattyacid ultra-free BSA fraction V (Roche Diagnostics Corporation,Indianapolis, Ind.). Data acquisition and processing was performed withan oocyte clamp OC-725B (Warner Instruments Corporation, Hamden, Conn.),pCLAMP (Axon, Sunnyvale, Calif.), IGOR Pro (WaveMetrics, Lake Oswego,Oreg.), and Origin6.1 (OriginLab Corporation, Northampton, Mass.)softwares. The equilibrium dissociation rates (K_(D)) for block ofKv1.1, Kv1.2, and Kv1.3 by mokatoxin-1 were determined by fitting thedose-response data and k_(on) and k_(off) were calculated as describedby Goldstein and Miller, 1993 (Biophys J. 1993 65 (4):1613-1619).

FIG. 16 and Table 11 show the dose-response relationship of mokatoxin-1on different K channels: human (h) Kv1.1, Kv1.2, Kv1.3, and mouse (m)big conductance calcium-activated K channel. The graph shows thatmokatoxin-1 is specific (selective) for Kv1.3 and has a high affinity.

TABLE 11 Mokatoxin-1 K_(on) AgTx-2 ChTx KTX Channel K_(i) [nM] [M · s]⁻¹K_(off) [s]⁻¹ K_(i) [nM] K_(i) [nM] K_(i) [nM] K_(on) [M · s]⁻¹ K_(off)[s]⁻¹ rK_(v)1.1 >>1 μM ND ND 0.17 ± 0.03 >>1 μM 1.1 ± 0.5 30 · 10⁶ ± 16· 10⁶ 0.009 ± 0.002 rK_(v)1.2 620 ± 60 0.26 · 10⁶ ± 0.06 · 10⁶ 0.16 ±0.03 3.4 ± 0.9 9 ± 4 20 ± 4  13 · 10⁶ ± 3 · 10⁶  0.21 ± 0.01 hK_(v)1.3 1.0 ± 0.1 36 · 10⁶ ± 5 · 10⁶  0.059 ± 0.007 0.041 ± 0.007 0.9 ± 0.10.008 ± 0.001 8 · 10⁸ ± 1 · 10⁸ 0.0070 ± 0.0009 mBK >>1 μM N/A N/A ND5.0 ± 0.8 1.6 ± 0.1 21 · 10³ ± 4 · 10³  0.0211 ± 0.0008

Example 14 Mokatoxin-1 Inhibits Human T Cell Function

Blocking the Kv1.3 channel in T cells suppresses immune responses. Datain this example show that mokatoxin-1 blocks T cell activity at least aswell as kaliotoxin. Experiments in this example were performed asdescribed in 2008, J. Biol. Chem. 283, 14559-14570.

Isolation and Culture of Human T Cells

Human T cells purified from peripheral blood of healthy donors.Peripheral blood mononuclear cells were separated by centrifugation overFicoll-Hypaque for 15 min. This step was followed by negative magneticcell purification according to the manufacturer's instructions (HumanPan T cells isolation kit, Miltenyi Biotec Inc., Auburn, Calif., USA)and purity of CD3⁺ T cells was >96% as assessed by flow cytometry. Cellswere maintained in RPMI 1640 medium supplemented with 10% FCS,penicillin, streptomycin, MOPS, 2-ME, and nonessential amino acids in a5% CO₂ incubator at 37° C.

IL-2, IFN-γ and TNF-α Secretion Assays

10⁵ freshly isolated CD3⁺ T cells were activated using CD3/CD28dynabeads (Dynal, USA) at a T cell:bead ratio of 1:1 in 200 ul of mediumin 96 wells plate. Channel blockers diluted at different concentrationsin PBS were added to the cells 1 h prior to stimulation (done intriplicate). After 16 h of activation cells were counted andsupernatants analysed for hIL-2, hIFN-γ and hTNF-α by ELISA followingthe manufacturer's instructions (eBiosciences, San Diego, Calif., USA).

Results: MK Toxin Inhibits Secretion of IL-2, IFN-γ and TNF-α byCD3/CD28 Activated T Cells

Block of the K⁺ channels in T cells by mokatoxin-1 decreased immunefunctions including IL-2 and IFN-γ secretion after CD3/CD28 stimulation.Human CD3⁺ T cells were stimulated with CD3/CD28 beads followingincubation with different concentrations (from 0.1 to 100 nM) of MK andKTX. The activity of MK was compared to the activity of KTX that waspreviously described to block the channels (Beeton et al, JI 2001). FIG.17 shows that MK inhibits at concentration as low as 1 nM. At aconcentration of 10 nM, IL-2 and TNF-α secretion were decreased byapproximately 50%; there was no reduction of cell viability withconcentrations as high as 1 μM (not shown). The data show thatinhibition of cytokine secretion by MK was comparable to, or better thanthat seen with KTX.

Examples 15 Mokatoxin-1 does not Alter Ileum Function

Kaliotoxin and related toxins block Kv1.1 and produce ilealcontractions. In contrast, mokatoxin is selective and does not blockKv1.1 or cause ileal hyperactivity.

To show the selectivity of mokatoxin-1 for Kv1.3 K⁺ channel subtype andto further demonstrate the pharmaceutical utility of mokatoxin-1,experiments were performed as described in Suarez-Kurtz et al., JPharmacol Exp Ther. 289 (3):1517-1222, 1999. These experiments use thespontaneous motility of guinea pig ileum as an organ-level assay todemonstrate K⁺ channel subtype specificity. The effect of Kv1.1 or Kv1.2blockers is to induce spontaneous twitches.

In addition, experiments were performed as described in Vianna-Jorge etal., Br J Pharmacol. 2003, 138 (1):57-62. These experiments assay theintraluminal pressure measurements of peristaltic activity of guinea pigileum as an organ-level assay to demonstrate K⁺ channel subtypespecificity. The effect of Kv1.1 or Kv1.2 blockers is to lower thepressure threshold for initiation of the peristaltic waves and increasethe frequency of these waves.

The classical kaliotoxin homolog Margatoxin at 10 nM, but notmokatoxin-1 (1-100 nM), induced twitches in the ileum strips (FIG. 18A).This is an effect mediated by Kv1.1 consistent with selectivity ofmokatoxin-1 for Kv1.3 channels (not in ileum). Margatoxin, but notmokatoxin-1 induced a lowering of the pressure threshold for initiationof the peristaltic waves and an increase in the frequency of these waves(FIG. 18B). Ileum has Kv1.1 and Kv1.2 channels and block of either leadsto contractions (as seen with all three toxins, margatoxin andkaliotoxin and agitoxin-2, J Pharmacol Exp Ther. 1999, 289(3):1517-1222). The effect by mokatoxin-1 on T cells but not ileum showsthat it blocks Kv1.3 but not Kv1.1 or Kv1.2 channels in native tissues.

Example 16 Mokatoxin-1 Structure is Novel

To understand the basis for selectivity the three dimensional structureof mokatoxin, the structure was solved by solution NMR by the methoddescribed in Koide et al., J Mol Biol. 284 (4):1141-1151, 1998; andKaratan et al., Chem Biol. 2004, 11 (6):835-844, 2004. This analysisrevealed that mokatoxin-1 and kaliotoxin-1 have similar, but notidentical, scaffold structure as expected by the constraints of librarydesign.

Example 17 Mokatoxin-2 and Mokatoxin-3: Kv Channel Blockers Selected onCells with Novel Characteristics

Mokatoxin-2 and -3 were isolated from a library using cell-based panningwhere CHO cells were induced to produce intact human Kv1.3 channels ontheir surfaces. (By contrast, mokatoxin-1 was isolated on purifiedproteins in plastic dishes). These novel toxins are based on sequencesfound in the kaliotoxin family and exhibit different kinetics ofinhibition compared to kaliotoxin-1, on which the library was designed,and to mokatoxin-1, a toxin peptide isolated using solid-phase panning.The amino acid sequences of mokatoxin-2 and mokatoxin-3 are as follows:

mokatoxin-2 (also known as mokatoxin_0422): (SEQ ID NO: _(——))TVIDVKCTSPKQCLPPCKAQFGIRAGAKCMNKKCRCYSmokatoxin-3 (also known as mokatoxin_0516): (SEQ ID NO: _(——))TVIDVKCTSPKQCLPPCKAQFGIRAGAKCMNKKCRCYS

To determine the kinetics of toxin block by these toxin peptides,oocytes were held at −100 mV and stepped to the test voltage of 0 mV for100 ms followed by a 200 ms duration step to −135 mV every 2 s. BKcurrents were recorded during a 50 ms step to +60 mV from a holdingvoltage of −80 mV, followed by a 40 ms step to −100 mV every 3 s. Theperfusion solution contained 2 mM KCl, 96 mM NaCl, 1 mM MgCl₂, 1.8 mMCaCl₂, 5 mM HEPES, pH 7.5 and 0.1% fatty acid ultra-free BSA fraction V(Roche Diagnostics Corporation, Indianapolis, Ind.). Data acquisitionand processing was performed with an oocyte clamp OC-725B (WarnerInstruments Corporation, Hamden, Conn.), pCLAMP (Axon, Sunnyvale,Calif.), IGOR Pro (WaveMetrics, Lake Oswego, Oreg.), and Origin6.1(OriginLab Corporation, Northampton, Mass.) softwares. The equilibriumdissociation rates (K_(D)) for block of Kv1.1, Kv1.2, and Kv1.3 bymokatoxins were determined by fitting the dose-response data and k_(on)and k_(off) were calculated as described by Goldstein and Miller, 1993(Biophys J. 1993 65 (4):1613-1619). The results are shown in FIGS. 19and 20.

Example 18 Phage Selection on Cells Allows for Screening of HighDiversity Libraries

After three rounds of panning, kaliotoxin-1-phage were recovered at 58%of recovered phage despite infrequent representation in the library.Phage expressing DDD-kaliotoxin, which is a mutant toxin that does notbind to KTX sites, do not bind to the channels. The data in Table 12show enrichment of kaliotoxin-1-phage (1 out of 10¹⁰ phage) on Kv1.3channels expressed in HEK cells. This shows the utility of thistechnique to select a highly diverse (e.g., 10¹⁰ or greater) library.

TABLE 12 2nd round 3rd round (n = 20) (n = 12) phage input proportion1st round % % kaliotoxin-1 0.0000000001 N.D.  0 58 DDD-kaliotoxin-10.9999999999 N.D. 100 42

Example 19 Toxins with Posttranslational Modifications and Generation ofDiversity

As described herein, libraries of ligands can be generated to includevariation by virtue of combinatorial diversity (e.g, by joining portionsof different toxins to create novel toxin peptide sequences) or sequencealterations. In some embodiments, diversity is generated by varyingresidues that undergo posttranslational modification.

Conus geographus GIIIA Libraries

This library is constructed using the marine cone snail Conus geographusGIIIA toxin as the scaffold. This toxin has amino acids that undergopostranslational modification in the snail (hydroxyproline, 0). In thisexample, residues that undergo postranslational modification are mutatedalone and in combination with other residues that are hypothesized togovern biological function to create lead toxins. Three examplelibraries and their resulting diversities are shown in Table 13:

* important for binding, based on literature and our own prediction− no effect on binding, based on literature and our own prediction

TABLE 13 Conus geographus GIIIA LibrariesRDCCTOOKKCKDRQCKOQRCCA* native toxin              * *  *     —       * —        — ** *             *  *             *  **             *        * *       * 0000000001111111111222 GIIIA residues1234567890123456789012 GIIIA residues.....123456789012..... mutagenesis residues RDCCTPPKKCKDRQCKPQRCCA-----XX---------X----- X = any amino acid except C library 1:diversity: approx. 19exp3 RDCCTPPKKCKDRQCKPQRCCA -----XX---------X-----library 2: diversity: approx. 185,193 RDCCTPPKKCKDRQCKPQRCCA-----XX-----H--RX-K---             K  H  H library 3:diversity: approx. 6.68 × 10exp7 RDCCTPPKKCKDRQCKPQRCCA-----XX-----HX-RXXK---             K  H  H

RDCCTOOKKCKDRQCKOQRCCA corresponds to SEQ ID NO:______.

Example 20 Representative Random Change Libraries

Shk-Scaffold Sea Anemone Library with Natural Variation at Key Sites

This library is constructed using Shk as the scaffold representativetoxin. All Shk scaffold member sequences are extracted from databases,literature, and related sources and aligned according to the disulfidebonds. In this example, only those Shk scaffold members are used wherethe number of amino acids between any of two neighboring cysteineresidues exactly match the number in Shk. Then a library design with acalculated diversity of 7.776000e+07 is produced. Other strategiesinclude use of representative residues for classes of amino acids, fullrandomization, etc.

Master Peptide: P29187/3-35 =RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC(SEQ ID NO: _(——)) Homologs: HmK (Heteractis magnifica)=RTCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC (SEQ ID NO: _(——))(Heteractis magnifica) =RTCKDLMPVSECTDIRCRTSMKYRLNLCRKTCGSC(SEQ ID NO: _(——)) AETX K =RACKDYLPKSECTQFRCRTSMKYKYTNCKKTCGTC(SEQ ID NO: _(——)) Q9TWG1/236 =..CKDNFAAATC....C..........CAKTCGKC(SEQ ID NO: _(——)) P81897/236 =..CKDNFSANTC....C..........CAKTCGKC(SEQ ID NO: _(——)) P29186/237 =..CRDWFKETAC....C..........CAKTCELC(SEQ ID NO: _(——)) Q8I9P4/11-46 =..C........C....C..........CAKTCGFC(SEQ ID NO: _(——)) A7S0M3/61-92 =..C........C....C..........CKKTCGTC(SEQ ID NO: _(——)) P29187/3-35 =..CIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC(SEQ ID NO: _(——)) A7S9H4/48-78 =..C........C....C..........CMKMCGLC(SEQ ID NO: _(——)) P11495/4-36 =..C........C....C..........CEKTC..C(SEQ ID NO: _(——)) A7T942/60-90 =..C........C....C..........CPKKCKYC(SEQ ID NO: _(——)) A7RS52/223-257 =..C........C....C..........CKKTCGHC(SEQ ID NO: _(——)) A7S9H4/283-313 =..C........C....C..........CPKKCGLC(SEQ ID NO: _(——)) A7RRU4/414-447 =..C........C....C..........CPRSCDYC(SEQ ID NO: _(——)) Q2MCX7/232-262 =..C........C....C..........CKKKCKLC(SEQ ID NO: _(——)) A7SMA6/73-119 =..C........C....C..........CAKTCGMC(SEQ ID NO: _(——)) A7T942/24-55 =..C........C....C..........CPKMCNYC(SEQ ID NO: _(——)) A7RW59/220-250 =..C........C....C..........CRKTCSLC(SEQ ID NO: _(——)) A7RW59/251-281 =..C........C....C..........CRKTCSLC(SEQ ID NO: _(——)) Q9TWG1/2-36 =..CKDNFAAATC....C..........CAKTCGKC(SEQ ID NO: _(——)) A7RZH9/186-226 =..C........C....C..........CAKTCGIC(SEQ ID NO: _(——)) A7T942/94-125 =..C........C....C..........CKKSCARC(SEQ ID NO: _(——)) Q9XZG0/458-496 =..C........C....CVSEEKTMKLYCRKTCNFC(SEQ ID —: _(——)) Q0MWV8/47-84 =..CSDRAHGHIC....C..........CKKTCGLC(SEQ ID NO: _(——)) P81897/2-36 =..CKDNFSANTC....C..........CAKTCGKC(SEQ ID NO: _(——)) A7SQ19/39-74 =..C........C....CRTNPKWMAKYCRKSCGTC(SEQ ID NO: _(——)) Q2MCX7/267-303 =..C........C....C..........CKRSCGLC(SEQ ID NO: _(——)) A7RMG1/3-35 =..C........C....C..........CQKSCDLC(SEQ ID NO: _(——)) A7RFK7/159-199 =..C........C....C..........CPETCGFC(SEQ ID NO: _(——)) A7RU87/237-269 =..C........C....C..........CKRSCKLC(SEQ ID NO: _(——)) P29186/2-37 =..CRDWFKETAC....C..........CAKTCELC(SEQ ID NO: _(——)) A7RNX2/29-63 =..C........C....CNKNPKWMLEHCRQSCGQC(SEQ ID NO: _(——)) A7RLA1/399-441 =..C........C....C..........CAKTCGYC(SEQ ID NO: _(——)) A7TC20/77-112 =..C........C....C..........CPKSCGIC(SEQ ID NO: _(——)) A7RVH8/171-211 =..C........C....C..........CLKSCGFC(SEQ ID NO: _(——)) A7SQK9/888-924 =..C........C....C..........CAYTCDTC(SEQ ID NO: _(——)) A7SCA9/62-99 =..C........C....C..........CGAACGLC(SEQ ID NO: _(——)) A7TC20/115-155 =..C........C....CQRNTKWMFHYCPVSCGIC(SEQ ID NO: _(——)) A7SMA5/494-532 =..C........C....C..........CKMTCNLC(SEQ ID NO: _(——)) A7SQK7/412-448 =..C........C....C..........CAYTCDTC(SEQ ID NO: _(——)) A7SY30/36-70 =..C........C....CTRNVKFMLDKCWRSCSGC(SEQ ID NO: _(——)) A7SKD2/68-105 =..C........C....C..........CAKSCAFC(SEQ ID NO: _(——)) A7SME3/290-326 =..C........C....C..........CRKTCSHC(SEQ ID NO: _(——)) Q9U4X9/193-232 =..C........C....C..........C...CKSC(SEQ ID NO: _(——)) A7T0S0/224-261 =..C........C....C..........CPKSCRMC(SEQ ID NO: _(——)) A7T1T5/33-71 =..C........C....C..........CQAACEIC(SEQ ID NO: _(——)) A7SV31/449-488 =..C........C....C..........CRRSCGSC(SEQ ID NO: _(——)) A7T7G9/127-163 =..C........C....C..........CKKSCNLC(SEQ ID NO: _(——)) A7S8T6/69-112 =..C........C....C..........CKKSCNLC(SEQ ID NO: _(——)) 0000000000000000000000000000000000000000000011111111112222222222333333 12345678901234567890123456789012345----------------------------------------------------------------------RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ..........E..DIR.RTEE.TKYNL..................T..Q...VSNP.WMKTN........ ..........A......NK.T.F.ALY..................I......QR.V...FKH........ .................T.......EK.................................H......... .........................D.........

1. A method of detecting the presence or absence of a ligand in a sample comprising: contacting at least one receptor with a sample comprising at least one toxin peptide; determining whether a toxin peptide in the sample selectively binds to the at least one receptor, thereby detecting the presence or absence of a ligand in a sample.
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. The method of claim 1, where the at least one receptor comprises a transmembrane protein.
 7. The method of claim 6, where the transmembrane protein is a channel protein.
 8. The method of claim 7, wherein the channel protein is a channel protein selected from the group consisting of a sodium ion channel, a potassium ion channel, a calcium ion channel, a chloride ion channel, a non-specific ion channel.
 9. The method of claim 8, where the transmembrane protein comprises a potassium ion channel.
 10. 10. The method of claim 9, where the transmembrane protein comprises a Kv1.3 channel.
 11. The method of claim 1, wherein the sample comprises a library of toxin peptides.
 12. The method of claim 11, where the library of toxin peptides comprises a phage display library.
 13. (canceled)
 14. (canceled)
 15. The method of claim 11, wherein the toxin peptides comprise the following amino acid sequence: (X)_(m)KC(X)_(n)QC(X)_(n)CK(X)_(o)KCM(X)_(n)CXC(X)_(m) (SEQ ID NO: 5), wherein X is any amino acid, and wherein m=0-10 amino acids, n=2-10 amino acids, and o=2-20 amino acids.
 16. (canceled)
 17. The method of claim 11, wherein the library comprises toxin peptides from one or more of a snake toxin, a snail toxin a scorpion toxin, a sea anemone toxin, and a lizard toxin.
 18. A library of toxin peptides, wherein members of the library comprise the following amino acid sequence: (X)_(m)KC(X)_(n)QC(X)_(n)CK(X)_(o)KCM(X)_(n)CXC(X)_(m) (SEQ ID NO: 5), wherein X is any amino acid, and wherein m=0-10 amino acids, n=2-10 amino acids, and o=2-20 amino acids.
 19. A library of toxin peptides, wherein the library comprises toxin peptides from one or more of a snake toxin, a snail toxin a scorpion toxin, a sea anemone toxin, and a lizard toxin.
 20. A peptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:
 1. 21. A peptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:
 2. 22. A peptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:
 3. 23. A pharmaceutical composition comprising the peptide of claim
 20. 24. A pharmaceutical composition comprising the peptide of claim
 21. 25. A pharmaceutical composition comprising the peptide of claim
 22. 26. A peptide comprising the following amino acid sequence: IXVKCXXPXQCXXPCKXXXGXXXXXKCMNXKCXCYX (SEQ ID NO: 7), wherein X is any amino acid, and wherein the peptide specifically binds to a potassium channel.
 27. A peptide comprising the following amino acid sequence: IXVKCXXPXQCXXPCKXXGXXXXKCMNXKCXCYX (SEQ ID NO: 8), wherein X is any amino acid, and wherein the peptide specifically binds to a potassium channel. 